Sequencing method employing ternary complex destabilization to identify cognate nucleotides

ABSTRACT

Provided are methods and systems for detecting formation of nucleotide-specific ternary complexes comprising a DNA polymerase, a nucleic acid, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide without requiring chemical incorporation of the nucleotide into the primer. These results can even be achieved in procedures employing unlabeled, native nucleotides.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/329,933, filed Apr. 29, 2016, and U.S. Provisional Patent ApplicationNo. 62/487,586, filed Apr. 20, 2017. The entire disclosures of theseearlier applications are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates generally to the field of biotechnology.More specifically, the invention concerns nucleic acid sequencingtechnology.

BACKGROUND

Acquiring accurate nucleic acid sequence information in a rapid andcost-effective manner is essential for the modern era of genomicanalysis. Certain automated DNA sequencing platforms require iterativecycles of enzyme-based nucleotide binding, incorporation into anextending primer, detection of incorporation reaction products, and evenchemical modification of the extended primer to render it useful in asubsequent cycle. Repeating the cycle for up to four candidatenucleotides to identify the cognate nucleotide at a single positionalong a DNA template complicates the workflow, and increases reagentcosts.

Stretches of more than one of the same base along a strand of nucleicacid are among factors confounding accurate sequence determination.These “homopolymer” stretches can be overlooked by some sequencingapproaches, such that a single base will be detected when multiplesactually are present. Some sequencing methods further can experience“phasing” issues that can be promoted by the presence of homopolymerstretches. As a consequence of phasing, sequence determinationdownstream of the homopolymer stretch can be rendered ambiguous.

Despite the many advances reported in the field of nucleic acidsequencing technology, there remains a need for improved systems thatdeliver accurate results quickly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an interferometry trace for interrogation of the first base incodon 12 of the KRAS wildtype (WT: GGT) and mutant (G12C: TGT; and G12R:CGT) sequences. Three different correct bases are shown at the sameposition for the three different template nucleic acids. The nextcorrect nucleotides harboring the correct bases are highlighted in bold.

FIG. 2 is an interferometry trace for interrogation of the second baseof codon 13 in KRAS WT (GGC) and G13D (GAC) sequences. Two differentcorrect bases are shown at the same position for the two differenttemplate nucleic acids. The next correct nucleotides harboring thecorrect bases are highlighted in bold.

FIGS. 3A-3D are interferometry traces for the double interrogationprotocol, wherein binary complexes formed between the primed templatenucleic acid molecule and polymerase were contacted with a plurality ofnucleotides in the absence of polymerase. The next correct nucleotidesharboring correct bases are highlighted in bold. The next correctnucleotide for the trace shown in FIG. 3A is dATP. The next correctnucleotide for the trace shown in FIG. 3B is dTTP. The next correctnucleotide for the trace shown in FIG. 3C is dGTP. The next correctnucleotide for the trace shown in FIG. 3D is dCTP.

FIGS. 4A-4B are interferometry traces for the examination andincorporation cycles of the expected sequence (GAC) in examplesequencing runs. Binding signals for all four dNTPs at the same positionare illustrated in the presence of 0.1 mM MgCl₂ (FIG. 4A) or 1 mM MgCl₂(FIG. 4B). All examination cycles were conducted after incorporating thecorrect 3′-blocked nucleotide, but before cleavage of the 3′-ONH₂reversible terminator moiety to reveal an extendable 3′-OH group. Basesfor the next correct incoming nucleotides (n+1) are highlighted usingbold uppercase base identifiers for their respective positions in thesequence. Additionally, the second correct bases (n+2) are highlightedusing bold lowercase base identifiers on their respective binding signalpeaks. The order of nucleotide examination was: dATP, dTTP, dGTP, anddCTP.

FIGS. 5A-5B are interferometry traces for the examination andincorporation cycles of an expected sequence (TGC), where bindingsignals for all four dNTPs are shown for a single interrogated positionin the presence of 1 mM MgCl₂. FIG. 5A presents results obtained usingBsu DNA polymerase in the examination steps. FIG. 5B presents resultsobtained using Bst 2.0 DNA polymerase in the examination steps. Allexamination cycles were conducted after incorporating the correctreversible terminator nucleotide, and before cleavage of 3′-ONH₂reversible terminator moiety of the primer to reveal an extendable 3′-OHgroup. Both figures display numerical indicators (1-8) of repetitiveprocess steps, with certain wash or regeneration steps therebetween: (1)incorporation of a reversible terminator nucleotide; (2) contacting withpolymerase in the absence of any nucleotide; (3) contacting with thecombination of polymerase, dATP, and dTTP; (4) contacting with thecombination of polymerase and dATP; (5) contacting with polymerase inthe absence of any nucleotide; (6) contacting with the combination ofpolymerase, dGTP, and dCTP; (7) contacting with the combination ofpolymerase and dCTP; and (8) chemical cleavage or removal of thereversible terminator moiety.

SUMMARY OF THE DISCLOSURE

In one aspect, the disclosure relates to a method of identifying anucleotide having a base complementary to the next base of a templatestrand immediately downstream of a primer in a primed template nucleicacid molecule. The method includes the steps of: (a) providing a blockedprimed template nucleic acid molecule including a reversible terminatormoiety that precludes the 3′-terminus of the blocked primed templatenucleic acid molecule from participating in phosphodiester bondformation; (b) contacting the blocked primed template nucleic acidmolecule with a first reaction mixture that includes a polymerase, and aplurality of different nucleotide molecules, whereby a stabilizedternary complex forms, the stabilized ternary complex including one ofthe plurality of different nucleotide molecules; (c) contacting thestabilized ternary complex with a second reaction mixture that includesat least one of the different nucleotide molecules and that does notinclude a first nucleotide molecule of the plurality of differentnucleotide molecules; (d) monitoring interaction of the polymerase andthe blocked primed template nucleic acid molecule in contact with thesecond reaction mixture to detect any of the stabilized ternary complexremaining after step (c); and (e) identifying the nucleotide thatincludes the base complementary to the next base of the template strandusing results from step (d). According to one generally preferredembodiment, the method further includes the step of (f) removing thereversible terminator moiety from the blocked primed template nucleicacid molecule after step (d). More preferably, step (e) can involvedetermining either that: (i) the first nucleotide molecule in step (c)includes the base complementary to the next base of the template strandif the stabilized ternary complex dissociates in step (d), or (ii) thefirst nucleotide molecule in step (c) does not include the basecomplementary to the next base of the template strand if the stabilizedternary complex is retained in step (d). Still more preferably, thepolymerase of the first reaction mixture can include an exogenousfluorescent label. Alternatively, the plurality of different nucleotidemolecules in the first reaction mixture can be either a plurality ofdifferent native nucleotide molecules, or a plurality of differentfluorescently labeled nucleotide molecules. When this is the case, thefirst reaction mixture may further include a catalytic metal ion.Alternatively, the first reaction mixture does not include non-catalyticmetal ions that inhibit phosphodiester bond formation by the polymeraseof the first reaction mixture, and the first reaction mixture furtherincludes a catalytic metal ion. According to other embodiments, wherethe method further includes the step of (f) removing the reversibleterminator moiety from the blocked primed template nucleic acid moleculeafter step (d), and where step (e) can involve determining either that:(i) the first nucleotide molecule in step (c) includes the basecomplementary to the next base of the template strand if the stabilizedternary complex dissociates in step (d), or (ii) the first nucleotidemolecule in step (c) does not include the base complementary to the nextbase of the template strand if the stabilized ternary complex isretained in step (d); step (a) can involve incorporating, with apolymerase, a reversible terminator nucleotide at the 3′-end of theprimer of the primed template nucleic acid molecule, whereby there isproduced the blocked primed template nucleic acid molecule including thereversible terminator moiety that precludes the 3′-terminus of theblocked primed template nucleic acid molecule from participating inphosphodiester bond formation. According to one preferred embodiment,the method further includes, after step (a) and before step (b), thestep of contacting the blocked primed template nucleic acid moleculewith the polymerase of the first reaction mixture in the absence theplurality of different nucleotide molecules. According to anotherpreferred embodiment, the second reaction mixture includes the samepolymerase that is present in the first reaction mixture. According toyet another preferred embodiment, the method further includes, afterstep (a) and before step (b), the step of contacting the blocked primedtemplate nucleic acid molecule with the polymerase of the first reactionmixture in the absence of the plurality of different nucleotidemolecules. For example, the first reaction mixture can further include acatalytic metal ion. Alternatively, the second reaction mixture caninclude the same polymerase that is present in the first reactionmixture. According to still other embodiments, where the method furtherincludes the step of (f) removing the reversible terminator moiety fromthe blocked primed template nucleic acid molecule after step (d), andwhere step (e) can involve determining either that: (i) the firstnucleotide molecule in step (c) includes the base complementary to thenext base of the template strand if the stabilized ternary complexdissociates in step (d), or (ii) the first nucleotide molecule in step(c) does not include the base complementary to the next base of thetemplate strand if the stabilized ternary complex is retained in step(d), and where step (a) involves incorporating, with a polymerase, areversible terminator nucleotide at the 3′-end of the primer of theprimed template nucleic acid molecule, whereby there is produced theblocked primed template nucleic acid molecule including the reversibleterminator moiety that precludes the 3′-terminus of the blocked primedtemplate nucleic acid molecule from participating in phosphodiester bondformation, the method can further involve repeating steps (b)-(e) aplurality of times. Here, the polymerase used in step (a) and thepolymerase of the first reaction mixture in step (b) can be differenttypes of polymerase enzymes. Alternatively, the polymerase of the firstreaction mixture includes an exogenous fluorescent label. Alternatively,the plurality of different nucleotide molecules in the first reactionmixture can be either a plurality of different native nucleotidemolecules, or a plurality of different fluorescently labeled nucleotidemolecules. Alternatively, the first reaction mixture further includes acatalytic metal ion. Alternatively, the first reaction mixture does notinclude non-catalytic metal ions that inhibit phosphodiester bondformation by the polymerase of the first reaction mixture.Alternatively, step (f) is performed before step (e). Alternatively, thesecond reaction mixture includes the same polymerase that is present inthe first reaction mixture.

In another aspect, the disclosure relates to a method of identifying anucleotide having a base complementary to the next base of a templatestrand immediately downstream of a primer in a primed template nucleicacid molecule. The method includes the steps of: (a) providing theprimed template nucleic acid molecule; (b) contacting the primedtemplate nucleic acid molecule with a first reaction mixture thatincludes a polymerase and a plurality of different nucleotide molecules,whereby a stabilized ternary complex forms, the stabilized ternarycomplex including one of the plurality of different nucleotidemolecules; (c) contacting the primed template nucleic acid molecule,after step (b), with a second reaction mixture that includes at leastone of the different nucleotide molecules and that does not include afirst nucleotide molecule of the plurality of different nucleotidemolecules; (d) monitoring interaction of the polymerase and the primedtemplate nucleic acid molecule in the second reaction mixture, withoutincorporating any nucleotide into the primer, to detect any of thestabilized ternary complex remaining after step (c); and (e) identifyingthe nucleotide that includes the base complementary to the next base ofthe template strand using results from step (d). According to onegenerally preferred embodiment, step (d) includes monitoring the rate ofdissociation of the polymerase from the primed template nucleic acidmolecule in the stabilized ternary complex. According to a differentgenerally preferred embodiment, the plurality of different nucleotidemolecules includes a plurality of different unlabeled nucleotidemolecules. More preferably, the polymerase of the first reaction mixtureincludes an exogenous fluorescent label producing a detectable signalthat is substantially unchanged in the presence or absence of the nextcorrect nucleotide. Alternatively, the first reaction mixture includesfour different types of native nucleotide molecules, and the secondreaction mixture does not include one of the four different types ofnative nucleotide molecules. Alternatively, the first reaction mixtureincludes two different types of native nucleotide molecules, and thesecond reaction mixture does not include one of the two different typesof native nucleotide molecules. Alternatively, the method furtherincludes an incorporation step that involves removing any of theplurality of different nucleotide molecules remaining in contact withthe primed template nucleic acid after step (d), contacting the primedtemplate nucleic acid with a third reaction mixture that includes apolymerase and at least one reversible terminator, and thenincorporating a single reversible terminator into the primer.Preferably, the polymerase of the first reaction mixture and thepolymerase of the third reaction mixture are different types of DNApolymerase, and the polymerase of the first reaction mixture includes anexogenous detectable label. More preferably, the exogenous detectablelabel includes a fluorescent label that is substantially unchanged inthe presence or absence of the next correct nucleotide. According toanother generally preferred embodiment, the method further includes anincorporation step that involves removing any of the plurality ofdifferent nucleotide molecules remaining in contact with the primedtemplate nucleic acid after step (d), contacting the primed templatenucleic acid with a third reaction mixture that includes a polymeraseand a nucleotide, and then incorporating the nucleotide of the thirdreaction mixture into the primer. According to another generallypreferred embodiment, the method further includes an incorporation stepthat involves removing any of the plurality of different nucleotidemolecules remaining in contact with the primed template nucleic acidafter step (d), contacting the primed template nucleic acid with a thirdreaction mixture that includes a polymerase and at least one reversibleterminator, and then incorporating a single reversible terminator intothe primer. According to generally preferred embodiments wherein themethod further includes an incorporation step that involves removing anyof the plurality of different nucleotide molecules remaining in contactwith the primed template nucleic acid after step (d), contacting theprimed template nucleic acid with a third reaction mixture that includesa polymerase and a nucleotide, and then incorporating the nucleotide ofthe third reaction mixture into the primer, the method can furtherinclude repeating each of steps (b)-(e) and the incorporation step.According to some embodiments, when the method further includes anincorporation step that involves removing any of the plurality ofdifferent nucleotide molecules remaining in contact with the primedtemplate nucleic acid after step (d), contacting the primed templatenucleic acid with a third reaction mixture that includes a polymeraseand at least one reversible terminator, and then incorporating a singlereversible terminator into the primer, the polymerase of the firstreaction mixture and the polymerase of the third reaction mixture can bedifferent types of DNA polymerase. More preferably, the polymerase ofthe first reaction mixture can include an exogenous fluorescent labelthat is not sensitive to nucleotide binding. According to someembodiments, when the method further includes an incorporation step thatinvolves removing any of the plurality of different nucleotide moleculesremaining in contact with the primed template nucleic acid after step(d), contacting the primed template nucleic acid with a third reactionmixture that includes a polymerase and at least one reversibleterminator, and then incorporating a single reversible terminator intothe primer, the at least one reversible terminator can include aplurality of different types of reversible terminators. More preferably,the plurality of different types of reversible terminators can includefour different reversible terminators. According to another generallypreferred embodiment, step (e) includes determining either that (i) thefirst nucleotide molecule includes the base complementary to the nextbase of the template strand if the stabilized ternary complexdissociates in step (d), or (ii) the first nucleotide molecule does notinclude the base complementary to the next base of the template strandif the stabilized ternary complex is retained in step (d). According toanother generally preferred embodiment, after step (b) and before step(c) there is a step (b)(i) that includes monitoring interaction of theprimed template nucleic acid molecule with the polymerase in the firstreaction mixture, without incorporating any nucleotides molecule intothe primer, to detect any of the stabilized ternary complex that formedin step (b). More preferably, step (e) involves determining that thefirst reaction mixture does not include the base complementary to thenext base of the template strand if the stabilized ternary complex wasnot detected in step (b)(i). According to another generally preferredembodiment, when step (d) includes monitoring the rate of dissociationof the polymerase from the primed template nucleic acid molecule in thestabilized ternary complex, and the plurality of different nucleotidemolecules include a plurality of different native nucleotide molecules,the method further includes an incorporation step. The incorporationstep can include removing any of the plurality of different nucleotidemolecules remaining in contact with the primed template nucleic acidafter step (d), contacting the primed template nucleic acid with a thirdreaction mixture that includes a polymerase and at least one reversibleterminator, and then incorporating a single reversible terminator intothe primer. Here, the polymerase of the first reaction mixture and thepolymerase of the third reaction mixture are different types of DNApolymerase.

In yet another aspect, the disclosure relates to a method of identifyinga nucleotide including a base complementary to the next base of atemplate strand immediately downstream of a primer in a primed templatenucleic acid molecule. The method includes the steps of: (a) providingthe primed template nucleic acid molecule; (b) contacting the primedtemplate nucleic acid molecule with a first reaction mixture thatincludes a polymerase, but does not include any nucleotide, whereby abinary complex forms; (c) contacting the binary complex with a secondreaction mixture that includes a plurality of different nucleotidemolecules, whereby a stabilized ternary complex forms if one of theplurality of different nucleotide molecules includes the basecomplementary to the next base of the template strand; (d) detecting,without incorporating any nucleotide into the primer, any of thestabilized ternary complex that may have formed; (e) contacting theprimed template nucleic acid molecule, after step (d), with a thirdreaction mixture that includes at least one of the different nucleotidemolecules and that does not include a first nucleotide molecule of theplurality of different nucleotide molecules; (f) detecting, withoutincorporating any nucleotide into the primer, any of the stabilizedternary complex remaining after step (e); and (g) identifying thenucleotide that includes the base complementary to the next base of thetemplate strand using results from both of detecting steps (d) and (f).According to one generally preferred embodiment, the method furtherincludes an incorporation step that involves first replacing the thirdreaction mixture in contact with the primed template nucleic acidmolecule with a fourth reaction mixture that includes a polymerase andat least one reversible terminator, and then incorporating the at leastone reversible terminator into the primer. According to a differentgenerally preferred embodiment, step (g) involves determining eitherthat (i) the first nucleotide molecule includes the base complementaryto the next base of the template strand if the stabilized ternarycomplex was detected in step (d) but was not detected in step (f), or(ii) the first nucleotide molecule does not include the basecomplementary to the next base of the template strand if the stabilizedternary complex was detected in both of steps (d) and (f), or (iii) thefirst reaction mixture does not include the nucleotide including thebase complementary to the next base of the template strand if thestabilized ternary complex was not detected in at least one of steps (d)and (f). More preferably, the method further includes an incorporationstep that involves first replacing the third reaction mixture in contactwith the primed template nucleic acid molecule with a fourth reactionmixture that includes a polymerase and at least one reversibleterminator, and then incorporating the at least one reversibleterminator into the primer. Alternatively, the method further includesan incorporation step that involves first replacing the third reactionmixture in contact with the primed template nucleic acid molecule with afourth reaction mixture that includes a polymerase and at least onereversible terminator, and then incorporating the at least onereversible terminator into the primer, and wherein the polymerase of thefirst reaction mixture and the polymerase of the fourth reaction mixtureare different types of DNA polymerase. When this is the case, steps(b)-(f) can be repeated two times using different nucleotides before theincorporation step is performed. According to a different generallypreferred embodiment, when step (g) involves determining either that (i)the first nucleotide molecule includes the base complementary to thenext base of the template strand if the stabilized ternary complex wasdetected in step (d) but was not detected in step (f), or (ii) the firstnucleotide molecule does not include the base complementary to the nextbase of the template strand if the stabilized ternary complex wasdetected in both of steps (d) and (f), or (iii) the first reactionmixture does not include the nucleotide including the base complementaryto the next base of the template strand if the stabilized ternarycomplex was not detected in at least one of steps (d) and (f), steps(b)-(f) can be repeated a plurality of times. According to a differentgenerally preferred embodiment, when step (g) involves determiningeither that (i) the first nucleotide molecule includes the basecomplementary to the next base of the template strand if the stabilizedternary complex was detected in step (d) but was not detected in step(f), or (ii) the first nucleotide molecule does not include the basecomplementary to the next base of the template strand if the stabilizedternary complex was detected in both of steps (d) and (f), or (iii) thefirst reaction mixture does not include the nucleotide including thebase complementary to the next base of the template strand if thestabilized ternary complex was not detected in at least one of steps (d)and (f), the primed template nucleic acid molecule of step (a) can beimmobilized to a surface. When this is the case, the primed templatenucleic acid molecule of step (a) can be immobilized to astreptavidin-coated surface. According to a different generallypreferred embodiment, when step (g) involves determining either that (i)the first nucleotide molecule includes the base complementary to thenext base of the template strand if the stabilized ternary complex wasdetected in step (d) but was not detected in step (f), or (ii) the firstnucleotide molecule does not include the base complementary to the nextbase of the template strand if the stabilized ternary complex wasdetected in both of steps (d) and (f), or (iii) the first reactionmixture does not include the nucleotide including the base complementaryto the next base of the template strand if the stabilized ternarycomplex was not detected in at least one of steps (d) and (f), step (g)can be performed by a computer programmed with software. According to adifferent generally preferred embodiment, when step (g) involvesdetermining either that (i) the first nucleotide molecule includes thebase complementary to the next base of the template strand if thestabilized ternary complex was detected in step (d) but was not detectedin step (f), or (ii) the first nucleotide molecule does not include thebase complementary to the next base of the template strand if thestabilized ternary complex was detected in both of steps (d) and (f), or(iii) the first reaction mixture does not include the nucleotideincluding the base complementary to the next base of the template strandif the stabilized ternary complex was not detected in at least one ofsteps (d) and (f), detecting steps (d) and (f) can involve opticaldetection. More preferably, detecting steps (d) and (f) can involvedetecting by interferometry. Alternatively, detecting steps (d) and (f)can involve detecting by surface plasmon resonance sensing. According toa different generally preferred embodiment, when step (g) involvesdetermining either that (i) the first nucleotide molecule includes thebase complementary to the next base of the template strand if thestabilized ternary complex was detected in step (d) but was not detectedin step (f), or (ii) the first nucleotide molecule does not include thebase complementary to the next base of the template strand if thestabilized ternary complex was detected in both of steps (d) and (f), or(iii) the first reaction mixture does not include the nucleotideincluding the base complementary to the next base of the template strandif the stabilized ternary complex was not detected in at least one ofsteps (d) and (f); and when the primed template nucleic acid molecule ofstep (a) is immobilized to a surface, step (c) can involve replacing thefirst reaction mixture with the second reaction mixture by flowing thesecond reaction mixture over the primed template nucleic acid moleculethat is immobilized to the surface. According to a different generallypreferred embodiment, when step (g) involves determining either that (i)the first nucleotide molecule includes the base complementary to thenext base of the template strand if the stabilized ternary complex wasdetected in step (d) but was not detected in step (f), or (ii) the firstnucleotide molecule does not include the base complementary to the nextbase of the template strand if the stabilized ternary complex wasdetected in both of steps (d) and (f), or (iii) the first reactionmixture does not include the nucleotide including the base complementaryto the next base of the template strand if the stabilized ternarycomplex was not detected in at least one of steps (d) and (f); and whenthe primed template nucleic acid molecule of step (a) is immobilized toa surface, step (c) can involve replacing the first reaction mixturewith the second reaction mixture by physically moving the primedtemplate nucleic acid molecule that is immobilized to the surface fromthe first reaction mixture to the second reaction mixture. According tostill yet another generally preferred embodiment, the primed templatenucleic acid molecule of step (a) is immobilized to a surface, andwherein step (d) includes either: replacing the first reaction mixturewith the second reaction mixture by flowing the second reaction mixtureover the primed template nucleic acid molecule that is immobilized tothe surface, or replacing the first reaction mixture with the secondreaction mixture by physically moving the primed template nucleic acidmolecule that is immobilized to the surface from the first reactionmixture to the second reaction mixture.

DETAILED DESCRIPTION

Disclosed is a technique for detecting ternary complexes that include aprimed template nucleic acid molecule, a polymerase, and the nextcorrect nucleotide immediately downstream of the primer andcomplementary to the template strand of a primed template nucleic acid.Clear and unambiguous detection has been achieved despite interactionsbetween the polymerase and the primed template nucleic acid that promoteformation of nucleotide-independent complexes.

The technique involves initial formation of a ternary complex using aplurality of nucleotides, and then subsequently investigating stabilityof the complex under a series of changed reagent conditions. Thesechanged conditions involve progressive removal of nucleotides from acontrolled series of binding reaction mixtures. For example, a ternarycomplex that includes a particular dNTP will require that dNTP in afirst reagent solution to maintain integrity of the complex. Exchangingthe first reagent solution with a second reagent solution that does notinclude the critical dNTP will cause destabilization of the complex,which can be detected as an indicator of nucleotide identity. Thisapproach permits a single incorporation reaction to be performed at theconclusion of multiple examinations, thereby reducing the number ofsteps and incorporation reagents needed to identify a single positionalong a primed template nucleic acid.

Advantageously, the technique can be practiced using various types ofnucleotides, including native (e.g., unlabeled) nucleotides, nucleotideswith detectable labels (e.g., fluorescent or other optically detectablelabels), or labeled or unlabeled nucleotide analogs (e.g., modifiednucleotides containing reversible terminator moieties). Further, thetechnique provides controlled reaction conditions, unambiguousdetermination of sequence, low overall cost of reagents, and lowinstrument cost.

The disclosed technique can be applied to binding reactions used fordetermining the identity of the next base of a primed template nucleicacid by any means and for any reason. The technique can be used tomonitor specific binding of a DNA polymerase and the next correctnucleotide (e.g., a dNTP) complementary to a primed template nucleicacid, and to distinguish specific binding from nonspecific binding. Thetechnique may be applied to single nucleotide determination (e.g., SNPdetermination), or alternatively to more extensive nucleic acidsequencing procedures employing reiterative cycles that identify onenucleotide at a time. For example, the methods provided herein can beused in connection with sequencing-by-binding procedures, as describedin the commonly owned U.S. patent application identified by Ser. No.14/805,381, the disclosure of which is incorporated by reference hereinin its entirety.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. For clarity, the following specific terms have the specifiedmeanings. Other terms are defined in other sections herein.

The singular forms “a” “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Approximating language, as usedin the description and claims, may be applied to modify any quantitativerepresentation that could permissibly vary without resulting in a changein the basic function to which it is related. Accordingly, a valuemodified by a term such as “about” is not to be limited to the precisevalue specified. Unless otherwise indicated, all numbers expressingquantities of ingredients, properties such as molecular weight, reactionconditions, so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the compositions, apparatus, or methods of thepresent disclosure. At the very least, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

As used herein, “sequencing-by-binding” refers to a sequencing techniquewherein specific binding of a polymerase to a primed template nucleicacid is used for identifying the next correct nucleotide to beincorporated into the primer strand of the primed template nucleic acid.The specific binding interaction precedes chemical incorporation of thenucleotide into the primer strand, and so identification of the nextcorrect nucleotide can take place either without or before incorporationof the next correct nucleotide.

As used herein, “nucleic acid” or “oligonucleotide” or “polynucleotide”or grammatical equivalents used herein means at least two nucleotidescovalently linked together. Thus, a “nucleic acid” is a polynucleotide,such as DNA, RNA, or any combination thereof, that can be acted upon bya polymerizing enzyme during nucleic acid synthesis. The term “nucleicacid” includes single-, double-, or multiple-stranded DNA, RNA andanalogs (derivatives) thereof. Double-stranded nucleic acidsadvantageously can minimize secondary structures that may hinder nucleicacid synthesis. A double stranded nucleic acid may possess a nick or asingle-stranded gap.

As used herein, a “template nucleic acid” is a nucleic acid to bedetected or sequenced using any sequencing method disclosed herein.

As used herein, a “primed template nucleic acid” (or alternatively,“primed template nucleic acid molecule”) is a template nucleic acidprimed with (i.e., hybridized to) a primer, wherein the primer is anoligonucleotide having a 3′-end with a sequence complementary to aportion of the template nucleic acid. The primer can optionally have afree 5′-end (e.g., the primer being noncovalently associated with thetemplate) or the primer can be continuous with the template (e.g., via ahairpin structure). The primed template nucleic acid includes thecomplementary primer and the template nucleic acid to which it is bound.Unless explicitly stated, a primed template nucleic acid can have eithera 3′-end that is extendible by a polymerase, or a 3′-end that is blockedfrom extension.

As used herein, a “blocked primed template nucleic acid” (oralternatively, “blocked primed template nucleic acid molecule”) is aprimed template nucleic acid modified to preclude or preventphosphodiester bond formation at the 3′-end of the primer. Blocking maybe accomplished, for example, by chemical modification with a blockinggroup at either the 3′ or 2′ position of the five-carbon sugar at the 3′terminus of the primer. Alternatively, or in addition, chemicalmodifications that preclude or prevent phosphodiester bond formation mayalso be made to the nitrogenous base of a nucleotide. Reversibleterminator nucleotide analogs including each of these types of blockinggroups will be familiar to those having an ordinary level of skill inthe art. Incorporation of these analogs at the 3′ terminus of a primerof a primed template nucleic acid molecule results in a blocked primedtemplate nucleic acid molecule. The blocked primed template nucleic acidincludes the complementary primer, blocked from extension at its 3′-end,and the template nucleic acid to which it is bound.

As used herein, a “nucleotide” is a molecule that includes a nitrogenousbase, a five-carbon sugar (ribose or deoxyribose), and at least onephosphate group. The term embraces, but is not limited to,ribonucleotides, deoxyribonucleotides, nucleotides modified to includeexogenous labels or reversible terminators, and nucleotide analogs.

As used herein, a “native” nucleotide refers to a naturally occurringnucleotide that does not include an exogenous label (e.g., a fluorescentdye, or other label) or chemical modification such as may characterize anucleotide analog. Examples of native nucleotides useful for carryingout the sequencing-by-binding procedures described herein include: dATP(2′-deoxyadenosine-5′-triphosphate); dGTP(2′-deoxyguanosine-5′-triphosphate); dCTP(2′-deoxycytidine-5′-triphosphate); dTTP(2′-deoxythymidine-5′-triphosphate); and dUTP(2′-deoxyuridine-5′-triphosphate).

As used herein, a “nucleotide analog” has one or more modifications,such as chemical moieties, which replace, remove and/or modify any ofthe components (e.g., nitrogenous base, five-carbon sugar, or phosphategroup(s)) of a native nucleotide. Nucleotide analogs may be eitherincorporable or non-incorporable by a polymerase in a nucleic acidpolymerization reaction. Optionally, the 3′-OH group of a nucleotideanalog is modified with a moiety. The moiety may be a 3′ reversible orirreversible terminator of polymerase extension. The base of anucleotide may be any of adenine, cytosine, guanine, thymine, or uracil,or analogs thereof. Optionally, a nucleotide has an inosine, xanthine,hypoxanthine, isocytosine, isoguanine, nitropyrrole (including3-nitropyrrole) or nitroindole (including 5-nitroindole) base.Nucleotides may include, but are not limited to, ATP, UTP, CTP, GTP,ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dUTP, dCTP, dGTP,dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Nucleotides may alsocontain terminating inhibitors of DNA polymerase, dideoxynucleotides or2′,3′ dideoxynucleotides, which are abbreviated as ddNTPs (ddGTP, ddATP,ddTTP, ddUTP and ddCTP).

As used herein, the “next template nucleotide” (or the “next templatebase”) refers to the next nucleotide (or base) in a template nucleicacid that is located immediately downstream of the 3′-end of ahybridized primer.

As used herein, the “next correct nucleotide” (sometimes referred to asthe “cognate” nucleotide) refers to the nucleotide type that will bindand/or incorporate at the 3′ end of a primer to complement a base in atemplate strand to which the primer is hybridized. The base in thetemplate strand is referred to as the “next template nucleotide” and isimmediately 5′ of the base in the template that is hybridized to the 3′end of the primer. The next correct nucleotide can be, but need notnecessarily be, capable of being incorporated at the 3′ end of theprimer. For example, the next correct nucleotide can be a member of aternary complex that will complete an incorporation reaction or,alternatively, the next correct nucleotide can be a member of astabilized ternary complex that does not catalyze an incorporationreaction. A nucleotide having a base that is not complementary to thenext template base is referred to as an “incorrect” (or “non-cognate”)nucleotide.

As used herein, a “blocking moiety,” when used with reference to anucleotide analog, is a part of the nucleotide that inhibits or preventsthe nucleotide from forming a covalent linkage to a second nucleotide(e.g., via the 3′-oxygen of a primer nucleotide) during theincorporation step of a nucleic acid polymerization reaction. Theblocking moiety of a “reversible terminator” nucleotide can be removedfrom the nucleotide analog to allow for nucleotide incorporation. Such ablocking moiety is referred to herein as a “reversible terminatormoiety.” Exemplary reversible terminator moieties are set forth in U.S.Pat Nos. 7,427,673; 7,414,116; and 7,057,026 and PCT publications WO91/06678 and WO 07/123744, each of which is incorporated by reference.

As used herein, a “test nucleotide” is a nucleotide being investigatedfor its ability to participate in formation of a ternary complex thatfurther includes a primed template nucleic acid and a polymerase.

As used herein, a “polymerase” is a generic term for a nucleic acidsynthesizing enzyme, including but not limited to, DNA polymerase, RNApolymerase, reverse transcriptase, primase and transferase. Typically,the polymerase includes one or more active sites at which nucleotidebinding and/or catalysis of nucleotide polymerization may occur. Thepolymerase may catalyze the polymerization of nucleotides to the 3′-endof a primer bound to its complementary nucleic acid strand. For example,a polymerase can catalyze the addition of a next correct nucleotide tothe 3′ oxygen of the primer via a phosphodiester bond, therebychemically incorporating the nucleotide into the primer. Optionally, thepolymerase used in the provided methods is a processive polymerase.Optionally, the polymerase used in the provided methods is adistributive polymerase. Optionally, a polymerase need not be capable ofnucleotide incorporation under one or more conditions used in a methodset forth herein. For example, a mutant polymerase may be capable offorming a ternary complex but incapable of catalyzing nucleotideincorporation.

As used herein, a “salt providing monovalent cation” is an ioniccompound that dissociates in aqueous solution to produce cations havinga single positive charge. For example, the cations can be metal cationswhere the oxidation state is +1.

As used herein, “a glutamate salt” is an ionic compound that dissociatesin aqueous solution to produce glutamate anions.

As used herein, “biphasic” refers to a two-stage process wherein aprimed template nucleic acid is contacted with a polymerase and a testnucleotide. The first phase of the process involves contacting theprimed template nucleic acid with a polymerase in the presence of asub-saturating level of nucleotide(s), or even in the absence ofnucleotides. The term “sub-saturating,” when used in reference to ligandthat binds to a receptor (e.g., a nucleotide that binds to apolymerase), refers to a concentration of the ligand that is below thatrequired to result in at least 90% of the receptors being bound to theligand at equilibrium. For example, a sub-saturating amount ofnucleotide can yield at least 90%, 95%, 99% or more polymerases beingbound to the nucleotide. The second phase of the process involvescontacting the primed template nucleic acid from the first phase with apolymerase in the presence of a higher concentration of nucleotide(s)than used in the first phase, where the higher concentration issufficient to yield maximal ternary complex formation when a nucleotidein the reaction is the next correct nucleotide.

As used herein, “providing” a template, a primer, a primed templatenucleic acid, or a blocked primed template nucleic acid refers to thepreparation and delivery of one or many nucleic acid polymers, forexample to a reaction mixture or reaction chamber.

As used herein, “monitoring” (or sometimes “measuring”) refers to aprocess of detecting a measurable interaction or binding between twomolecular species. For example, monitoring may involve detectingmeasurable interactions between a polymerase and primed template nucleicacid, typically at various points throughout a procedure. Monitoring canbe intermittent (e.g., periodic) or continuous (e.g., withoutinterruption), and can involve acquisition of quantitative results.Monitoring can be carried out by detecting multiple signals over aperiod of time during a binding event or, alternatively, by detectingsignal(s) at a single time point during or after a binding event.

As used herein, the term “solid support” refers to a rigid substratethat is insoluble in aqueous liquid. The substrate can be non-porous orporous. The substrate can optionally be capable of taking up a liquid(e.g., due to porosity) but will typically be sufficiently rigid thatthe substrate does not swell substantially when taking up the liquid anddoes not contract substantially when the liquid is removed by drying. Anonporous solid support is generally impermeable to liquids or gases.Exemplary solid supports include, but are not limited to, glass andmodified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™,cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor,silica or silica-based materials including silicon and modified silicon,carbon, metals, inorganic glasses, optical fiber bundles, and polymers.

As used herein, “contacting” refers to the mixing together of reagents(e.g., mixing an immobilized template nucleic acid and either a bufferedsolution that includes a polymerase, or the combination of a polymeraseand a test nucleotide) so that a physical binding reaction or a chemicalreaction may take place.

As used herein, “incorporating” or “chemically incorporating” refers tothe process of joining a cognate nucleotide to a primer by formation ofa phosphodiester bond.

As used herein, a “binary complex” is an intermolecular associationbetween a polymerase and a primed template nucleic acid (or blockedprimed template nucleic acid), where the complex does not include anucleotide molecule such as the next correct nucleotide.

As used herein, a “ternary complex” is an intermolecular associationbetween a polymerase, a primed template nucleic acid (or blocked primedtemplate nucleic acid), and the next correct nucleotide positionedimmediately downstream of the primer and complementary to the templatestrand of the primed template nucleic acid or the blocked primedtemplate nucleic acid. The primed template nucleic acid can include, forexample, a primer with a free 3′-OH or a blocked primer (e.g., a primerwith a chemical modification on the base or the sugar moiety of the 3′terminal nucleotide, where the modification precludes enzymaticphosphodiester bond formation). The term “stabilized ternary complex”means a ternary complex having promoted or prolonged existence or aternary complex for which disruption has been inhibited. Generally,stabilization of the ternary complex prevents covalent incorporation ofthe nucleotide component of the ternary complex into the primed nucleicacid component of the ternary complex.

As used herein, a “catalytic metal ion” refers to a metal ion thatfacilitates phosphodiester bond formation between the 3′-OH of a nucleicacid (e.g., a primer) and the phosphate of an incoming nucleotide by apolymerase. A “divalent catalytic metal cation” is a catalytic metal ionhaving a valence of two. Catalytic metal ions can be present atconcentrations necessary to stabilize formation of a complex between apolymerase, a nucleotide, and a primed template nucleic acid, referredto as non-catalytic concentrations of a metal ion. Catalyticconcentrations of a metal ion refer to the amount of a metal ionsufficient for polymerases to catalyze the reaction between the 3′-OHgroup of a nucleic acid (e.g., a primer) and the phosphate group of anincoming nucleotide.

As used herein, a “non-catalytic metal ion” refers to a metal ion that,when in the presence of a polymerase enzyme, does not facilitatephosphodiester bond formation needed for chemical incorporation of anucleotide into a primer. Typically, the non-catalytic metal ion is acation. A non-catalytic metal ion may inhibit phosphodiester bondformation by a polymerase, and so may stabilize a ternary complex bypreventing nucleotide incorporation. Non-catalytic metal ions mayinteract with polymerases, for example, via competitive binding comparedto catalytic metal ions. A “divalent non-catalytic metal ion” is anon-catalytic metal ion having a valence of two. Examples of divalentnon-catalytic metal ions include, but are not limited to, Ca²⁺, Zn²⁺,Co²⁺, Ni²⁺, and Sr²⁺. The trivalent Eu³⁺ and Tb³⁺ ions are non-catalyticmetal ions having a valence of three.

As used herein an “exogenous label” refers to a detectable chemicalmoiety that has been added to a sequencing reagent, such as a nucleotideor a polymerase (e.g., a DNA polymerase).

While a native dNTP may have a characteristic limited fluorescenceprofile, the native dNTP does not include any added colorimetric orfluorescent moiety. Conversely, a dATP(2′-deoxyadenosine-5′-triphosphate) molecule modified to include achemical linker and fluorescent moiety attached to the gamma phosphatewould be said to include an exogenous label because the attachedchemical components are not ordinarily a part of the nucleotide. Ofcourse, chemical modifications to add detectable labels to nucleotidebases also would be considered exogenous labels. Likewise, a DNApolymerase modified to include a conformationally sensitive fluorescentdye that changes its properties upon nucleotide binding also would besaid to include an exogenous label because the label is not ordinarily apart of the polymerase.

As used herein, “unlabeled” refers to a molecular species free of addedor exogenous label(s) or tag(s). Of course, unlabeled nucleotides willnot include either of an exogenous fluorescent label, or an exogenousRaman scattering tag. A native nucleotide is another example of anunlabeled molecular species. An unlabeled molecular species can excludeone or more of the labels set forth herein or otherwise known in the artrelevant to nucleic acid sequencing or analytical biochemistry.

Sequencing-By-Binding

Described herein are polymerase-based, nucleic acidsequencing-by-binding (SBB) reactions, wherein the polymerase undergoesconformational transitions between open and closed conformations duringdiscrete steps of the reaction. In one step, the polymerase binds to aprimed template nucleic acid to form a binary complex, also referred toherein as the pre-insertion conformation. In a subsequent step, anincoming nucleotide is bound and the polymerase fingers close, forming apre-chemistry conformation including a polymerase, primed templatenucleic acid and nucleotide; wherein the bound nucleotide has not beenincorporated. This step, also referred to herein as the “examination”step, may be followed by a chemical step wherein a phosphodiester bondis formed with concomitant pyrophosphate cleavage from the nucleotide(i.e., nucleotide incorporation). The polymerase, primed templatenucleic acid and newly incorporated nucleotide produce a post-chemistry,pre-translocation conformation. As both the pre-chemistry conformationand the pre-translocation conformation include a polymerase, primedtemplate nucleic acid and nucleotide, wherein the polymerase is in aclosed state, either conformation may be referred to herein as aclosed-complex or a closed ternary complex. In the closed pre-insertionstate, divalent catalytic metal ions, such as Mg²⁺ mediate a rapidchemical reaction involving nucleophilic displacement of a pyrophosphate(PPi) by the 3′ hydroxyl of the primer. The polymerase returns to anopen state upon the release of PPi, the post-translocation step, andtranslocation initiates the next round of reaction. While aclosed-complex can form in the absence of divalent catalytic metal ions(e.g., Mg²⁺), the polymerase of the closed complex is proficient inchemical addition of nucleotide in the presence of the divalent metalions when provided with an appropriate substrate having an available3′hydroxyl group. Low or deficient levels of catalytic metal ions, suchas Mg²⁺, lead to non-covalent (e.g., physical) sequestration of the nextcorrect nucleotide in a closed-complex. This closed-complex may bereferred to as a stabilized or trapped closed-complex. In any reactionstep described above, the polymerase configuration and/or interactionwith a nucleic acid may be monitored during an examination step toidentify the next correct base in the template nucleic acid sequence.Before or after incorporation, reaction conditions can be changed todisengage the polymerase from the primed template nucleic acid, andchanged again to remove from the local environment any reagents thatinhibit polymerase binding.

Generally speaking, the SBB procedure includes an “examination” stepthat identifies the next template base, and optionally an“incorporation” step that adds one or more complementary nucleotides tothe 3′-end of the primer component of the primed template nucleic acid.Identity of the next correct nucleotide to be added is determined eitherwithout, or before chemical linkage of that nucleotide to the 3′-end ofthe primer through a covalent bond. The examination step can involveproviding a primed template nucleic acid to be used in the procedure,and contacting the primed template nucleic acid with a polymerase enzyme(e.g., a DNA polymerase) and one or more test nucleotides beinginvestigated as the possible next correct nucleotide. Further, there isa step that involves monitoring or measuring the interaction between thepolymerase and the primed template nucleic acid in the presence of thetest nucleotides. Optionally, the interaction can take place in thepresence of stabilizers, whereby the polymerase-nucleic acid interactionis stabilized in the presence of the next correct nucleotide. Again, theexamination step identifies or determines the identity of the nextcorrect nucleotide without requiring incorporation of that nucleotide.Stated differently, identity of the next correct nucleotide can beestablished without chemical incorporation of the nucleotide into theprimer when one or more cycles of examination is carried out usinglabeled or unlabeled nucleotides.

Whereas methods involving a single template nucleic acid molecule may bedescribed for convenience, these methods are exemplary. The sequencingmethods provided herein readily encompass a plurality of templatenucleic acids, wherein the plurality of nucleic acids may be clonallyamplified copies of a single nucleic acid, or disparate nucleic acids,including combinations, such as populations of disparate nucleic acidsthat are clonally amplified. Thus, such sequencing methods are fullydisclosed herein.

The Examination Step

An examination step according to the technique described hereintypically includes the following substeps: (1) providing a primedtemplate nucleic acid (i.e., a template nucleic acid molecule hybridizedwith a primer that optionally may be blocked from extension at its3′-end); (2) contacting the primed template nucleic acid with a reactionmixture that includes a polymerase and at least one nucleotide; (3)monitoring the interaction of the polymerase with the primed templatenucleic acid molecule in the presence of the nucleotide(s) and withoutchemical incorporation of any nucleotide into the primed templatenucleic acid; and (4) identifying the next base in the template nucleicacid (i.e., the next correct nucleotide) using the monitoredinteraction. Optionally, the primed template nucleic acid molecule canbe contacted initially with the polymerase in the absence ofnucleotide(s) before contacting any nucleotide. The primer of the primedtemplate nucleic acid can be an extendible primer. Alternatively, theprimer of the primed template nucleic acid is blocked from extension atits 3′-end. The primed template nucleic acid, the polymerase and thetest nucleotide are capable of forming a ternary complex when the baseof the test nucleotide is complementary to the next base of the primedtemplate nucleic acid molecule. Under some conditions the primedtemplate nucleic acid and the polymerase may be capable of forming abinary complex when the base of the test nucleotide is not complementaryto the next base of the primed template nucleic acid molecule.Optionally, the contacting occurs under conditions that favor formationof the ternary complex over formation of the binary complex. Theidentifying step can include identifying the base of the nucleotide thatis complementary to the next base of the primed template nucleic acid.Optionally, this includes contacting ternary complexes with one or morewash solutions having different nucleotide compositions that permitternary complexes to be selectively maintained or dissociated.

All of these steps can be repeated one or more times to obtain extensivesequence information. For example, ternary complexes can be formedinitially by contacting a primed template nucleic acid (optionallyincluding a blocked 3′-end) with a polymerase (optionally labeled withan exogenous label) and a plurality of nucleotides (optionally includingone or more exogenous labels). Solution conditions can be changed suchthat ternary complexes are contacted with a wash solution that includesonly a subset of nucleotides used for forming the ternary complex.Optionally, this solution includes the same polymerase used to form theternary complex. Monitoring interaction of the polymerase and/ornucleotide in the ternary complex can be carried out to determinewhether the ternary complex remains stable (thereby indicating that oneof the nucleotides in the wash buffer corresponds to the cognatenucleotide) or becomes destabilized (thereby indicating that the bufferno longer contains the cognate nucleotide). The wash steps can berepeated until the ternary complex becomes destabilized (e.g., to thepoint of dissociating) by progressively omitting one nucleotide that waspresent during the preceding wash cycle. Optionally, a cognatenucleotide can be incorporated following one or a plurality of reagentexchanges.

All of these steps can be repeated one or more times to obtain extensivesequence information. For example, the contacting and monitoring stepscan be repeated one or more times. Optionally, the contacting andmonitoring steps are repeated using a reaction mixture that includes thepolymerase and a first test nucleotide. Optionally, the contacting andmonitoring steps are repeated using a reaction mixture that includes thepolymerase and a second nucleotide. Optionally, the contacting andmonitoring steps are repeated using a reaction mixture that includes thepolymerase and a third nucleotide. Optionally, the contacting andmonitoring steps are repeated using a reaction mixture that includes thepolymerase and a fourth nucleotide.

In the sequencing methods provided herein, the reaction mixture used forforming ternary complexes, that includes the DNA polymerase and at leastone test nucleotide, can include at least 1, 2, 3, or 4 types ofnucleotide molecules (e.g., either labeled or unlabeled nucleotides).Optionally, the nucleotides are native nucleotides selected from dATP,dTTP, dCTP, and dGTP. Optionally, the reaction mixture includes one ormore triphosphate nucleotides and one or more diphosphate nucleotides.Optionally, the polymerase includes a detectable label (e.g., afluorescent label). Optionally, any fluorescent label joined to anucleotide or a polymerase is not an intercalating dye, a conformationaldye, a FRET partner, or other label that substantially changesfluorescent emission as a consequence of participating in a binary orternary complex, or participating in binding of nucleotide topolymerase. Optionally, a closed-complex is formed between the primedtemplate nucleic acid, the polymerase, and one of four nucleotidemolecules included in the reaction mixture. Localization of detectablelabel to the position of the primed template nucleic acid (e.g., a“nucleic acid feature” on a solid support, such as a microarray) isdetected and used for deducing cognate and/or non-cognate nucleotideidentity.

In a particular example of the provided method, the primed templatenucleic acid (optionally blocked from extension at its 3′-end) iscontacted with a reaction mixture that includes polymerase with one ormore nucleotides. A ternary complex will form if one or more of thenucleotides is a cognate nucleotide for the position being interrogated.

In another particular example of the provided method, the primedtemplate nucleic acid (optionally blocked at its 3′-end) is initiallycontacted with a reaction mixture that includes polymerase without addedtest nucleotide. Thereafter, the primed template nucleic acid iscontacted with a reaction mixture that includes polymerase and one ormore test nucleotides that may participate in ternary complex formation.Thereafter, the optionally blocked primed template nucleic acid iscontacted with a reaction mixture that includes polymerase and one fewernucleotide than the preceding reaction mixture. Monitoring maintenanceor destabilization of any ternary complex can take place continuously,or after each reaction mixture change.

The examination step may be controlled so that nucleotide incorporationis either attenuated or accomplished. If nucleotide incorporation isattenuated during the examination step, then a separate incorporationstep may be performed after determining the identity of the next correctnucleotide. The separate incorporation step may be accomplished withoutthe need for monitoring, as the cognate nucleotide has already beenidentified during the examination step. If nucleotide incorporationproceeds during examination, subsequent nucleotide incorporation may beattenuated by use of a stabilizer that traps the polymerase on thenucleic acid after incorporation. A reversibly terminated nucleotide mayalso be used to prevent the addition of subsequent nucleotides. The SBBmethod allows for controlled determination of a template nucleic acidbase without requiring the use of labeled nucleotides, as theinteraction between the polymerase and template nucleic acid can bemonitored without a label on the nucleotide. To be clear, however, theuse of a labeled nucleotide (e.g., a fluorescent nucleotide) is optionalwhen performing the presently disclosed procedure to allow forfluorescent detection of bound nucleotide.

In the sequencing methods provided herein, the test nucleotide (e.g., atleast one test nucleotide) can include a 3′ hydroxyl group, or ablocking moiety that prevents phosphodiester bond formation at the3′-end of the primer. A 3′ terminator moiety or a 2′ terminator moietymay be either a reversible terminator or an irreversible terminator.Optionally, the reversible terminator of the at least one nucleotidemolecule is replaced or removed at some point after the examination stepthat employed the test nucleotide that included the reversibleterminator.

Contacting Steps

Contacting of the primed template nucleic acid molecule with reactionmixtures that include the polymerase and one or more test nucleotidemolecules can occur under conditions that stabilize formation of theternary complex and/or destabilize formation of the binary complex.Optionally, the reaction mixture includes potassium glutamate.Optionally, the conditions that stabilize formation of the ternarycomplex include contacting the primed template nucleic acid with astabilizing agent. Optionally, the reaction mixture includes astabilizing agent. The stabilizing agent can be one or morenon-catalytic metal ions that inhibit polymerase incorporation.Exemplary non-catalytic metal ions include calcium ion, strontium ion,tin ion, nickel ion, and europium ion. For example, the reaction mixtureof the examination step that includes the primed template nucleic acid,the polymerase, and the test nucleotide also may include from 0.01 mM to30 mM strontium chloride as a stabilizing agent.

Alternatively, and particularly when using a blocked primed templatenucleic acid to form a ternary complex in the examination step, reactionmixtures used for conducting examination and monitoring steps optionallycan include catalytic metal ions (e.g., Mg²⁺ or Mn²⁺). Concentrations ofthe catalytic metal ions needed to support polymerization activity whenusing unmodified (i.e., not 3′ blocked) primers will be familiar tothose having an ordinary level of skill in the art.

In certain embodiments, the primed template nucleic acid is immobilizedto the surface of a solid support. The immobilization may employ eithera covalent or a noncovalent bond between one or the other, or even bothstrands of the primed template nucleic acid and the solid support. Forexample, when the template and primer strands of the primed templatenucleic acid are different molecules, the template strand can beimmobilized, for example via its 5′-end. What is necessary, however, isthat the 3′ terminus of the primer is available for interacting with thepolymerase, whether the 3′-end is extendible or blocked from extensionby a polymerase.

When the primed template nucleic acid is immobilized to a solid support,there are alternatives for how the contacting steps are performed. Forexample, the solid support can be physically transferred betweendifferent vessels (e.g., individual wells of a multiwell plate)containing different reagent solutions. This is convenientlyaccomplished using an automated or robotic instrument. In anotherexample, the primed template nucleic acid is immobilized to a solidsupport inside a flow cell or chamber. In this instance, differentcontacting steps can be executed by controlled flow of different liquidreagents through the chamber, or across the immobilized primed templatenucleic acid.

The Monitoring Step

Monitoring or measuring the interaction of the polymerase with theprimed template nucleic acid molecule in the presence of a nucleotidemolecule may be accomplished in many different ways. For example,monitoring can include measuring association kinetics for theinteraction between the primed template nucleic acid, the polymerase,and a nucleotide. Monitoring the interaction of the polymerase with theprimed template nucleic acid molecule in the presence of a nucleotidemolecule can include measuring equilibrium binding constants between thepolymerase and primed template nucleic acid molecule (i.e., equilibriumbinding constants of polymerase to the template nucleic acid in thepresence of a nucleotide). Thus, for example, the monitoring includesmeasuring the equilibrium binding constant of the polymerase to theprimed template nucleic acid in the presence of a nucleotide. Monitoringthe interaction of the polymerase with the primed template nucleic acidmolecule in the presence of a nucleotide molecule includes measuringdissociation kinetics of the polymerase from the primed template nucleicacid in the presence of any one of the four nucleotides. Optionally,monitoring the interaction of the polymerase with the primed templatenucleic acid molecule in the presence of a nucleotide molecule includesmeasuring kinetics of the dissociation of the closed-complex (i.e.,dissociation of the primed template nucleic acid, the polymerase, andthe nucleotide). Optionally, the measured association kinetics differdepending on the identity of the nucleotide molecule. Optionally, thepolymerase has a different affinity for each type of nucleotideemployed. Optionally, the polymerase has a different dissociationconstant for each type of nucleotide in each type of closed-complex.Association, equilibrium and dissociation kinetics are known and can bereadily determined by one in the art. See, for example, Markiewicz etal., Nucleic Acids Research 40(16):7975-84 (2012); Xia et al., J. Am.Chem. Soc. 135(1):193-202 (2013); Brown et al., J. Nucleic Acids,Article ID 871939, 11 pages (2010); Washington, et al.,

Mol. Cell. Biol. 24(2):936-43 (2004); Walsh and Beuning, J. NucleicAcids, Article ID 530963, 17 pages (2012); and Roettger, et al.,Biochemistry 47(37):9718-9727 (2008), which are incorporated byreference herein in their entireties.

The monitoring step can include monitoring the steady state interactionof the polymerase with the primed template nucleic acid in the presenceof a first nucleotide, without chemical incorporation of the firstnucleotide into the primer of the primed template nucleic acid.Optionally, monitoring includes monitoring the dissociation of thepolymerase from the primed template nucleic acid in the presence of afirst nucleotide, without chemical incorporation of the first nucleotideinto the primer of the primed template nucleic acid. Optionally,monitoring includes monitoring the association of the polymerase withthe primed template nucleic acid in the presence of the firstnucleotide, without chemical incorporation of the first nucleotide intothe primer of the primed template nucleic acid. Again, test nucleotidesin these procedures may be native nucleotides (i.e., unlabeled), labelednucleotides (e.g., fluorescently labeled nucleotides), or nucleotideanalogs (e.g., nucleotides modified to include reversible orirreversible terminator moieties).

In some aspects of the sequencing methods provided herein, a reversiblyblocked primer prevents the chemical incorporation of the nucleotideinto the primer of the primed template nucleic acid. This stabilizes anyternary complex that may have formed. Optionally, a catalytic metal ion(e.g., magnesium ion) is present in the examination reaction mixturethat includes the reversibly blocked primed template nucleic acidmolecule.

In other aspects of the sequencing methods provided herein, the absenceof a catalytic metal ion in the reaction mixture or the absence of acatalytic metal ion in the active site of the polymerase prevents thechemical incorporation of the nucleotide into the primer of the primedtemplate nucleic acid. Optionally, the chelation of a catalytic metalion in the reaction mixtures of the contacting step prevents thechemical incorporation of the nucleotide into the primer of the primedtemplate nucleic acid. Optionally, a non-catalytic metal ion acts as astabilizer for the ternary closed-complex in the presence of the nextcorrect nucleotide. Optionally, the substitution of a catalytic metalion in the reaction mixtures of the contacting step with a non-catalyticmetal ion prevents the chemical incorporation of the nucleotide moleculeto the primed template nucleic acid. Optionally, the catalytic metal ionis magnesium. The metal ion mechanisms of polymerases postulates that alow concentration of metal ions may be needed to stabilize thepolymerase-nucleotide-DNA binding interaction. See, for instance,Section 27.2.2, Berg J M, Tymoczko J L, Stryer L, Biochemistry 5thEdition, WH Freeman Press, 2002.

Optionally, a low concentration of a catalytic ion in the reactionmixtures of the examination step (i.e., that are used for bindingpolymerase in the presence or absence of a test nucleotide) prevents thechemical incorporation of the test nucleotide into the primer of theprimed template nucleic acid. Optionally, a low concentration of thecatalytic ion (e.g., magnesium ion) is from about 1 μM to less than 100μM. Optionally, a low concentration is from about 0.5 μM to about 5 μM.Optionally, the reaction mixtures of the examination step includecobalt, and the incorporating step includes contacting with anincorporation reaction mixture containing a higher concentration ofcobalt as compared to the concentration of cobalt in the reactionmixtures of the examination step.

The examination step may be controlled, in part, by providing reactionconditions to prevent chemical incorporation of a nucleotide whileallowing monitoring of the interaction between the polymerase and theprimed template nucleic acid, thereby permitting determination of theidentity of the next base of the nucleic acid template strand. Suchreaction conditions may be referred to as “examination reactionconditions.” Optionally, a ternary complex or closed-complex is formedunder examination conditions. Optionally, a stabilized ternary complexor closed-complex is formed under examination conditions or in apre-chemistry conformation. Optionally, a stabilized closed-complex isin a pre-translocation conformation, wherein the enclosed nucleotide hasbeen incorporated, but the closed-complex does not allow for theincorporation of a subsequent nucleotide. Optionally, the examinationconditions accentuate the difference in affinity for polymerase toprimed template nucleic acids in the presence of different nucleotides.Optionally, the examination conditions cause differential affinity ofthe polymerase to the primed template nucleic acid in the presence ofdifferent nucleotides. By way of example, the examination conditionsthat cause differential affinity of the polymerase to the primedtemplate nucleic acid in the presence of different nucleotides include,but are not limited to, high salt and inclusion of potassium glutamate.Concentrations of potassium glutamate that can be used to alterpolymerase affinity for the primed template nucleic acid include 10 mMto 1.6 M of potassium glutamate, or any amount in between 10 mM and 1.6M. Optionally, high salt refers to a concentration of salt from 50 mM to1,500 mM salt. Preferably, the salt is a salt providing monovalentcations.

Examination typically involves, in the monitoring step, detectingpolymerase interaction with a template nucleic acid, or with templatenucleic acid and nucleotide in combination. Detection may includeoptical, electrical, thermal, acoustic, chemical and mechanical means.Optionally, monitoring is performed after a buffer change or a washstep, wherein the wash step removes any non-bound reagents (e.g.,unbound polymerases and/or nucleotides) from the region of observation.Optionally, monitoring is performed during a buffer change or a washstep, such that the dissociation kinetics of the polymerase-nucleic acidor polymerase-nucleic acid-nucleotide complexes may be used to determinethe identity of the next base. Optionally, monitoring is performedduring the course of addition of the examination reaction mixture orfirst reaction mixture, such that the association kinetics of thepolymerase to the nucleic acid may be used to determine the identity ofthe next base on the nucleic acid. Optionally, monitoring involvesdistinguishing closed-complexes from binary complexes of polymerase andprimed template nucleic acid. Optionally, monitoring is performed underequilibrium conditions where the affinities measured are equilibriumaffinities. Multiple examination steps including different or similarexamination reagents, may be performed sequentially to ascertain theidentity of the next template base. Multiple examination steps may beutilized in cases where multiple template nucleic acids are beingsequenced simultaneously in one sequencing reaction, wherein differentnucleic acids react differently to the different examination reagents.Optionally, multiple examination steps may improve the accuracy of nextbase determination.

In an exemplary sequencing reaction, the examination step includesformation and/or stabilization of a closed-complex including apolymerase, a primed template nucleic acid, and the next correctnucleotide. Characteristics of the formation and/or release of theclosed-complex are monitored to identify the enclosed nucleotide andtherefore the next base in the template nucleic acid. Closed-complexcharacteristics can be dependent on the sequencing reaction components(e.g., polymerase, primer, template nucleic acid, nucleotide) and/orreaction mixture components and/or conditions. Optionally, theclosed-complex is in a pre-chemistry conformation. Optionally, theclosed-complex is in a pre-translocation conformation. Optionally, theclosed-complex is in a post-translocation conformation.

The examination step involves monitoring the interaction of a polymerasewith a primed template nucleic acid in the presence of a testnucleotide. In some embodiments, this can involve monitoring theinteraction of a detectably labeled polymerase with the primed templatenucleic acid. In other embodiments, this can involve monitoring adetectable signal (e.g., a fluorescent emission) produced by adetectably labeled test nucleotide. In still other embodiments, thesystem is a label-free system based on monitoring binding of unlabeledpolymerase to a surface (e.g., using surface plasmon resonance sensing,or interferometry). The formation of a closed-complex may be monitored.Optionally, the absence of formation of a closed-complex is monitored.Optionally, the dissociation of a closed-complex is monitored.Optionally, the incorporation step involves monitoring incorporation ofa nucleotide. Optionally, the incorporation step involves monitoring theabsence of nucleotide incorporation.

Any process of the examination and/or incorporation step may bemonitored. Optionally, a polymerase has an exogenous label or “tag.”Optionally, the detectable tag or label on the polymerase is removable.Optionally, the nucleotide harbors a detectable label (e.g., acovalently attached fluorescent label). Optionally, the nucleotides orpolymerases have a detectable label, however, the label is not detectedduring sequencing. Optionally, no component of the sequencing reactionis detectably labeled with an exogenous label.

Monitoring the variation in affinity of a polymerase for a templatenucleic acid in the presence of correct and incorrect nucleotides, underconditions that may or may not allow the incorporation of thenucleotide, may be used to determine the sequence of the nucleic acid.The affinity of a polymerase for a template nucleic acid in the presenceof different nucleotides, including modified or labeled nucleotides, canbe monitored as the off-rate of the polymerase-nucleic acid interactionin the presence of the various nucleotides. The affinities and off-ratesof many standard polymerases to various matched/correct,mismatched/incorrect and modified nucleotides are known in the art.Single molecule imaging of Klenow polymerase reveals that the off-ratefor a template nucleic acid for different nucleotide types, where thenucleotide types are prevented from incorporating, are distinctly andmeasurably different.

Optionally, a nucleotide of a particular type is made available to apolymerase in the presence of a primed template nucleic acid. Thereaction is monitored, wherein, if the nucleotide is a next correctnucleotide, the polymerase may be stabilized to form a closed-complex.If the nucleotide is an incorrect nucleotide, a closed-complex may stillbe formed; however, without the additional assistance of stabilizingagents or reaction conditions (e.g., absence of catalytic ions,polymerase inhibitors, salt), the closed-complex may dissociate. Therate of dissociation is dependent on the affinity of the particularcombination of polymerase, template nucleic acid, and nucleotide, aswell as reaction conditions. Optionally, the affinity is measured as anoff-rate. Optionally, the affinity is different between differentnucleotides for the closed-complex. For example, if the next base in thetemplate nucleic acid downstream of the 3′-end of the primer is G, thepolymerase-nucleic acid affinity, measured as an off-rate, is expectedto be different based on whether dATP, dCTP, dGTP or dTTP are added. Inthis case, dCTP would have the slowest off-rate, with the othernucleotides providing different off-rates for the interaction.Optionally, the off-rate may be different depending on the reactionconditions, for example, the presence of stabilizing agents (e.g.,absence of magnesium or inhibitory compounds) or reaction conditions(e.g., nucleotide modifications or modified polymerases). Once theidentity of the next correct nucleotide is determined, 1, 2, 3, 4 ormore nucleotide types may be introduced simultaneously to the reactionmixture under conditions that specifically target the formation of aclosed-complex. Excess nucleotides may be removed from the reactionmixture and the reaction conditions modulated to incorporate the nextcorrect nucleotide of the closed-complex. This sequencing reactionensures that only one nucleotide is incorporated per sequencing cycle.

The affinity of a polymerase for a template nucleic acid in the presenceof a nucleotide can be measured in a plurality of methods known to oneof skill in the art. Optionally, the affinity is measured as anoff-rate, where the off-rate is measured by monitoring the release ofthe polymerase from the template nucleic acid as the reaction is washedby a wash buffer. Optionally, the affinity is measured as an off-rate,where the off-rate is measured by monitoring the release of thepolymerase from the template nucleic acid under equilibrium bindingconditions, especially equilibrium binding conditions in which thepolymerase binding rates are low or diffusion limited. The polymerasebinding rates may be diffusion limited at sufficiently lowconcentrations of polymerase, wherein if the polymerase falls off fromthe DNA-polymerase complex, it does not load back immediately, therebyallowing for sufficient time to detect that the polymerase has beenreleased from the complex. For a higher affinity interaction, thepolymerase is released from the nucleic acid slowly, whereas a lowaffinity interaction results in the polymerase being released morerapidly. The spectrum of affinities, in this case, translates todifferent off-rates, with the off-rates measured under dynamic washconditions or at equilibrium. The smallest off-rate corresponds to thebase complementary to the added nucleotide, while the other off-ratesvary, in a known fashion, depending on the combination of polymerase andnucleotide selected.

Optionally, the off-rate is measured as an equilibrium signal intensityafter the polymerase and nucleotide are provided in the reactionmixture, wherein the interaction with the lowest off-rate (highestaffinity) nucleotide produces the strongest signal, while theinteractions with other, varying, off-rate nucleotides produce signalsof measurably different intensities. As a non-limiting example, afluorescently labeled polymerase, measured, preferably, under totalinternal reflection (TIRF) conditions, produces different measuredfluorescence intensities depending on the number of polymerase moleculesbound to surface-immobilized nucleic acid molecules in a suitably chosenwindow of time. The intrinsic fluorescence of the polymerase, forinstance, tryptophan fluorescence, may also be utilized. A high off-rateinteraction produces low measured intensities, as the number of boundpolymerase molecules, in the chosen time window is very small, wherein ahigh off-rate indicates that most of the polymerase is unbound from thenucleic acid.

Any surface localized measurement scheme may be employed including, butnot limited to, labeled or fluorescence schemes. Suitable measurementschemes that measure affinities under equilibrium conditions include,but are not limited to, bound mass, refractive index, surface charge,dielectric constant, and other schemes known in the art. Optionally, acombination of on-rate and off-rate engineering yields higher fidelitydetection in the proposed schemes. As a non-limiting example, auniformly low on-rate, base-dependent, varying off-rate results in anunbound polymerase remaining unbound for prolonged periods, allowingenhanced discrimination of the variation in off-rate and measuredintensity. The on-rate may be manipulated by lowering the concentrationof the added polymerase, nucleotide, or both polymerase and nucleotide.

Optionally, the interaction between the polymerase and the nucleic acidis monitored via a detectable tag attached to the polymerase. The tagmay be monitored by detection methods including, but limited to,optical, electrical, thermal, mass, size, charge, vibration, andpressure. The label may be magnetic, fluorescent or charged. Forexternal and internal label schemes, fluorescence anisotropy may be usedto determine the stable binding of a polymerase to a nucleic acid in aclosed-complex.

By way of example, a polymerase is tagged with a fluorophore, whereinclosed-complex formation is monitored as a stable fluorescent signal.The unstable interaction of the polymerase with the template nucleicacid in the presence of an incorrect nucleotide results in a measurablyweaker signal compared to the closed-complex formed in the presence ofthe next correct nucleotide. In certain preferred embodiments, however,the sequencing-by-binding procedure does not rely on detection of anyexogenous label (e.g., a fluorescent label) joined to the polymerase.For example, the polymerase can be a native polymerase.

Optionally, a primed template nucleic acid molecule (optionally blockedat its 3′-end) is contacted with polymerase and one or more exogenouslylabeled nucleotides during the examination step. Monitoring of signalgenerated as a consequence of the presence of the labeled nucleotideprovides information concerning formation andstabilization/destabilization of the ternary complex that includes thelabeled nucleotide. For example, if the exogenous label is a fluorescentlabel, and if the primed template nucleic acid is immobilized to a solidsupport at a particular locus, then monitoring fluorescent signalassociated with that locus can be used for monitoring ternary complexformation and stability under different reaction mixture conditions.

The Identifying Step

The identity of the next correct base or nucleotide can be determined bymonitoring the presence, formation and/or dissociation of the ternarycomplex or closed-complex. The identity of the next base may bedetermined without chemically incorporating the next correct nucleotideinto the 3′-end of the primer. Optionally, the identity of the next baseis determined by monitoring the affinity of the polymerase for theprimed template nucleic acid in the presence of added nucleotides.Optionally, the affinity of the polymerase for the primed templatenucleic acid in the presence of the next correct nucleotide may be usedto determine the next correct base on the template nucleic acid.Optionally, the affinity of the polymerase for the primed templatenucleic acid in the presence of an incorrect nucleotide may be used todetermine the next correct base on the template nucleic acid.

In certain embodiments, a ternary complex that includes a primedtemplate nucleic acid (or a blocked primed template nucleic acid) isformed in the presence of a polymerase and a plurality of nucleotides.Cognate nucleotide participating in the ternary complex optionally isidentified by observing destabilization of the complex that occurs whenthe cognate nucleotide is absent from the reaction mixture. This isconveniently carried out, for example, by exchanging one reactionmixture for another. Here, loss of the complex is an indicator ofcognate nucleotide identity. Loss of binding signal (e.g., a fluorescentbinding signal associated with a particular locus on a solid support)can occur when the primed template nucleic acid is exposed to a reactionmixture that does not include the cognate nucleotide. Optionally,maintenance of a ternary complex in the presence of a single nucleotidein a reaction mixture also can indicate identity of the cognatenucleotide.

The Incorporation Step

Optionally, the methods provided herein further include an incorporationstep. By way of example, the incorporation step includes incorporating asingle nucleotide (e.g., an unlabeled nucleotide, a reversibleterminator nucleotide, or a detectably labeled nucleotide analog)complementary to the next base of the template nucleic acid into theprimer of the primed template nucleic acid molecule. Optionally, theincorporation step includes contacting the primed template nucleic acidmolecule, polymerase and nucleotide with an incorporation reactionmixture. The incorporation reaction mixture, typically includes acatalytic metal ion.

The provided method may further include preparing the primed templatenucleic acid molecule for a next examination step after theincorporation step. Optionally, the preparing includes subjecting theprimed template nucleic acid or the nucleic acid/polymerase complex toone or more wash steps; a temperature change; a mechanical vibration; apH change; salt or buffer composition changes, an optical stimulation ora combination thereof. Optionally, the wash step includes contacting theprimed template nucleic acid or the primed template nucleicacid/polymerase complex with one or more buffers, detergents, proteindenaturants, proteases, oxidizing agents, reducing agents, or otheragents capable of releasing internal crosslinks within a polymerase orcrosslinks between a polymerase and nucleic acid.

Optionally, the method further includes repeating the examination stepand the incorporation step to sequence a template nucleic acid molecule.The examination step may be repeated one or more times prior toperforming the incorporation step. Optionally, two consecutiveexamination steps include reaction mixtures with different nucleotidemolecules (e.g., different nucleotides that are labeled or unlabeled).Optionally, prior to incorporating the single nucleotide into the primedtemplate nucleic acid molecule, the first reaction mixture is replacedwith a second reaction mixture including a polymerase and 1, 2, 3, or 4types of nucleotide molecules (e.g., different unlabeled nucleotides).Optionally, the nucleotide molecules are native nucleotides selectedfrom dATP, dTTP, dCTP, and dGTP.

The incorporation reaction may be enabled by an incorporation reactionmixture. Optionally, the incorporation reaction mixture includes adifferent composition of nucleotides than the examination reaction. Forexample, the examination reaction includes one type of nucleotide andthe incorporation reaction includes another type of nucleotide. By wayof another example, the examination reaction includes one type ofnucleotide and the incorporation reaction includes four types ofnucleotides, or vice versa. Optionally, the examination reaction mixtureis altered or replaced by the incorporation reaction mixture.Optionally, the incorporation reaction mixture includes a catalyticmetal ion, potassium chloride, or a combination thereof.

Nucleotides present in the reaction mixture but not sequestered in aclosed-complex may cause multiple nucleotide insertions. Thus, a washstep can be employed prior to the chemical incorporation step to ensureonly the nucleotide sequestered within a trapped closed-complex isavailable for incorporation during the incorporation step. Optionally,free nucleotides may be removed by enzymes such as phosphatases. Thetrapped polymerase complex may be a closed-complex, a stabilizedclosed-complex or ternary complex involving the polymerase, primedtemplate nucleic acid and next correct nucleotide.

Optionally, the nucleotide enclosed within the closed-complex of theexamination step is incorporated into the 3′-end of the template nucleicacid primer during the incorporation step. Optionally, the nucleotideenclosed within the closed-complex of the examination step isincorporated during the examination step, but the closed-complex doesnot allow for the incorporation of a subsequent nucleotide; in thisinstance, the closed-complex is released during an incorporation step,allowing for a subsequent nucleotide to become incorporated.

Optionally, the incorporation step includes replacing a nucleotide fromthe examination step and incorporating another nucleotide into the3′-end of the template nucleic acid primer. The incorporation step canfurther involve releasing a nucleotide from within a closed-complex(e.g., the nucleotide is a modified nucleotide or nucleotide analog) andincorporating a nucleotide of a different kind to the 3′-end of thetemplate nucleic acid primer. Optionally, the released nucleotide isremoved and replaced with an incorporation reaction mixture including anext correct nucleotide.

Suitable reaction conditions for incorporation may involve replacing theexamination reaction mixture with an incorporation reaction mixture.Optionally, nucleotides present in the examination reaction mixture arereplaced with one or more nucleotides in the incorporation reactionmixture. Optionally, the polymerase present during the examination stepis replaced during the incorporation step. Optionally, the polymerasepresent during the examination step is modified during the incorporationstep. Optionally, the one or more nucleotides present during theexamination step are modified during the incorporation step. Thereaction mixture and/or reaction conditions present during theexamination step may be altered by any means during the incorporationstep. These means include, but are not limited to, removing reagents,chelating reagents, diluting reagents, adding reagents, alteringreaction conditions such as conductivity or pH, and any combinationthereof. The reagents in the reaction mixture including any combinationof polymerase, primed template nucleic acid, and nucleotide may bemodified during the examination step and/or incorporation step.

Optionally, the reaction mixture of the incorporation step includescompetitive inhibitors, wherein the competitive inhibitors reduce theoccurrence of multiple incorporations. In certain embodiments, thecompetitive inhibitor is a non-incorporable nucleotide. In certainembodiments, the competitive inhibitor is an aminoglycoside. Thecompetitive inhibitor is capable of replacing either the nucleotide orthe catalytic metal ion in the active site, such that after the firstincorporation the competitive inhibitor occupies the active sitepreventing a second incorporation. In some embodiments, both anincorporable nucleotide and a competitive inhibitor are introduced inthe incorporation step, such that the ratio of the incorporablenucleotide and the inhibitor can be adjusted to ensure incorporation ofa single nucleotide at the 3′-end of the primer.

Optionally, the provided reaction mixtures, including the incorporationreaction mixtures, include at least one unlabeled nucleotide moleculethat is a non-incorporable nucleotide. In other words, the providedreaction mixtures can include one or more unlabeled nucleotide moleculesthat are incapable of incorporation into the primer of the primedtemplate nucleic acid molecule. Nucleotides incapable of incorporationinclude, for example, diphosphate nucleotides. For instance, thenucleotide may contain modifications to the triphosphate group that makethe nucleotide non-incorporable. Examples of non-incorporablenucleotides may be found in U.S. Pat. No. 7,482,120, the disclosure ofwhich is incorporated by reference herein in its entirety. Optionally,the primer may not contain a free hydroxyl group at its 3′-end, therebyrendering the primer incapable of incorporating any nucleotide, and,thus making any nucleotide non-incorporable.

A polymerase inhibitor optionally may be included with the reactionmixtures containing test nucleotides in the examination step to trap thepolymerase on the nucleic acid upon binding the next correct nucleotide.Optionally, the polymerase inhibitor is a pyrophosphate analog.Optionally, the polymerase inhibitor is an allosteric inhibitor.Optionally, the polymerase inhibitor is a DNA or an RNA aptamer.Optionally, the polymerase inhibitor competes with a catalytic-ionbinding site in the polymerase. Optionally, the polymerase inhibitor isa reverse transcriptase inhibitor. The polymerase inhibitor may be anHIV-1 reverse transcriptase inhibitor or an HIV-2 reverse transcriptaseinhibitor. The HIV-1 reverse transcriptase inhibitor may be a(4/6-halogen/MeO/EtO-substituted benzo[d]thiazol-2-yl)thiazolidin-4-one.

In the provided sequencing methods, the next correct nucleotide isidentified before the incorporation step, allowing the incorporationstep to not require labeled reagents and/or monitoring. Thus, in theprovided methods, a nucleotide, optionally, does not contain an attacheddetectable tag or label. Optionally, the nucleotide contains adetectable label, but the label is not detected in the method.Optionally, the correct nucleotide does not contain a detectable label;however, an incorrect or non-complementary nucleotide to the next basecontains a detectable label.

The examination step of the sequencing reaction may be repeated 1, 2, 3,4 or more times prior to the incorporation step. The examination andincorporation steps may be repeated until the desired sequence of thetemplate nucleic acid is obtained.

The formation of the closed-complex or the stabilized closed-complex canbe employed to ensure that only one nucleotide is added to the templatenucleic acid primer per cycle of sequencing, wherein the addednucleotide is sequestered within the closed-complex. The controlledincorporation of a single nucleotide per sequencing cycle enhancessequencing accuracy for nucleic acid regions including homopolymerrepeats. Optionally, only reversible terminator nucleotides areincorporated into an extendible primer by the action of a polymeraseover the course of several cognate nucleotide identification cycles. Thereversible terminator nucleotides can be unlabeled reversible terminatornucleotides (e.g., having 3′-ONH₂ reversible terminator moieties)

Reaction Mixtures

Nucleic acid sequencing reaction mixtures, or simply “reactionmixtures,” typically include reagents that are commonly present inpolymerase-based nucleic acid synthesis reactions. Reaction mixturereagents include, but are not limited to, enzymes (e.g., thepolymerase), dNTPs, template nucleic acids, primer nucleic acids, salts,buffers, small molecules, co-factors, metals, and ions. The ions may becatalytic ions, divalent catalytic ions, non-catalytic ions,non-covalent metal ions, or a combination thereof. The reaction mixturecan include salts such as NaCl, KCl, potassium acetate, ammoniumacetate, potassium glutamate, NH₄Cl, or NH₄HSO₄. The reaction mixturecan include a source of ions, such as Mg²⁺ or Mn²⁺ Mg-acetate, Co²⁺ orBa²⁺. The reaction mixture can include tin ions, Ca²⁺, Zn²⁺, Cu²⁺, Co²⁺,Fe²⁺, Ni²⁺, or Eu⁺³. The buffer can include Tris, Tricine, HEPES, MOPS,ACES, MES, phosphate-based buffers, and acetate-based buffers. Thereaction mixture can include chelating agents such as EDTA, EGTA, andthe like. Optionally, the reaction mixture includes cross-linkingreagents. Provided herein are reaction mixtures, optionally, used duringthe examination step, as well as incorporation reaction mixtures usedduring nucleotide incorporation that can include one or more of theaforementioned agents. Reaction mixtures, when used during examination,can be referred to herein as examination reaction mixtures. Optionally,the examination reaction mixture includes a high concentration of salt(e.g., a salt providing monovalent cations); a high pH; 1, 2, 3, 4, ormore types of unlabeled nucleotides; potassium glutamate; a chelatingagent; a polymerase inhibitor; a catalytic metal ion; a non-catalyticmetal ion; or any combination thereof. The examination reaction mixturecan include 10 mM to 1.6 M of potassium glutamate or any amount inbetween 10 mM and 1.6 M. Optionally, the incorporation reaction mixtureincludes a catalytic metal ion; 1, 2, 3, 4, or more types of nucleotides(e.g., unlabeled nucleotides); potassium chloride; a non-catalytic metalion; or any combination thereof.

Optionally, reaction mixtures in accordance with the disclosedtechniques modulate the formation and stabilization of a closed-complexduring an examination step. For example, the reaction conditions of theexamination step optionally can favor the formation and/or stabilizationof a closed-complex encapsulating a nucleotide, and hinder the formationand/or stabilization of a binary complex. The binary interaction betweenthe polymerase and template nucleic acid may be manipulated bymodulating sequencing reaction parameters such as ionic strength, pH,temperature, or any combination thereof, or by the addition of a binarycomplex destabilizing agent to the reaction. Optionally, high salt(e.g., 50 mM to 1,500 mM) and/or pH changes are utilized to destabilizea binary complex. Optionally, the salt used for providing the high saltconditions is a salt that provides monovalent cations. Optionally, abinary complex may form between a polymerase and a template nucleic acidduring the examination or incorporation step of the sequencing reaction,regardless of the presence of a nucleotide. Optionally, the reactionconditions favor the stabilization of a closed ternary complex anddestabilization of a binary complex. By way of example, the pH of theexamination reaction mixture can be adjusted from pH 4.0 to pH 10.0 tofavor the stabilization of a closed ternary complex and destabilizationof a binary complex. Optionally, the pH of the examination reactionmixture is from pH 4.0 to pH 6.0. Optionally, the pH of the examinationreaction mixture is pH 6.0 to pH 10.0.

The provided reaction mixtures and sequencing methods disclosed hereinencourage polymerase interaction with the nucleotides and templatenucleic acid in a manner that reveals the identity of the next basewhile controlling the chemical addition of a nucleotide. Optionally, themethods are performed in the absence of detectably labeled nucleotidesor in the presence of labeled nucleotides wherein the labels are notdetected. Optionally, the reaction mixtures include nucleotides thatharbor an exogenous detectable label (e.g., a fluorescent label).Optionally, a plurality of nucleotides in a reaction mixture harbor thesame exogenous detectable label. Optionally, a plurality of nucleotidesin a reaction mixture harbor different exogenous detectable labels.Optionally, the reaction mixtures can include one or more exogenouslylabeled polymerase enzymes.

Provided herein are reaction mixtures and methods that facilitateformation and/or stabilization of a closed-complex that includes apolymerase bound to a primed template nucleic acid and a nucleotideenclosed within the polymerase-template nucleic acid complex, underexamination reaction mixture conditions. Examination reaction conditionsmay inhibit or attenuate nucleotide incorporation. Optionally,incorporation of the enclosed nucleotide is inhibited and the complex isstabilized or trapped in a pre-chemistry conformation or a ternarycomplex. Optionally, the enclosed nucleotide is incorporated andsubsequent nucleotide incorporation is inhibited. In this instance, thecomplex is stabilized or trapped in a pre-translocation conformation.For the sequencing reactions provided herein, the closed-complex isstabilized during the examination step, allowing for controllednucleotide incorporation. Optionally, a stabilized closed-complex is acomplex wherein incorporation of an enclosed nucleotide is attenuated,either transiently (e.g., to examine the complex and then incorporatethe nucleotide) or permanently (e.g., for examination only) during anexamination step. Optionally, a stabilized closed-complex allows for theincorporation of the enclosed nucleotide, but does not allow for theincorporation of a subsequent nucleotide. Optionally, the closed-complexis stabilized in order to monitor any polymerase interaction with atemplate nucleic acid in the presence of a nucleotide for identificationof the next base in the template nucleic acid.

Optionally, the enclosed nucleotide has severely reduced or disabledbinding to the template nucleic acid in the closed-complex. Optionally,the enclosed nucleotide is base-paired to the template nucleic acid at anext base. Optionally, the identity of the polymerase, nucleotide,primer, template nucleic acid, or any combination thereof, affects theinteraction between the enclosed nucleotide and the template nucleicacid in the closed-complex.

Optionally, the enclosed nucleotide is bound to the polymerase of theclosed-complex. Optionally, the enclosed nucleotide is weakly associatedwith the polymerase of the closed-complex. Optionally, the identity ofthe polymerase, nucleotide, primer, template nucleic acid, or anycombination thereof, affects the interaction between the enclosednucleotide and the polymerase in the closed-complex. For a givenpolymerase, each nucleotide has a different affinity for the polymerasethan another nucleotide. Optionally, this affinity is dependent, inpart, on the template nucleic acid and/or the primer.

The closed-complex may be transiently formed. Optionally, the enclosednucleotide is a next correct nucleotide. In some methods, the presenceof the next correct nucleotide contributes, in part, to thestabilization of a closed-complex. Optionally, the enclosed nucleotideis not a next correct nucleotide.

Optionally, the examination reaction condition comprises a plurality ofprimed template nucleic acids, polymerases, nucleotides, or anycombination thereof. Optionally, the plurality of nucleotides comprises1, 2, 3, 4, or more types of different nucleotides, for example dATP,dTTP, dGTP, and dCTP. Optionally, the plurality of template nucleicacids is a clonal population of template nucleic acids.

Reaction conditions that may modulate the stability of a closed-complexinclude, but are not limited to, the availability of catalytic metalions, suboptimal or inhibitory metal ions, ionic strength, pH,temperature, polymerase inhibitors, cross-linking reagents, the presenceor absence of a reversible terminator moiety on the 3′ nucleotide of theprimed template nucleic acid molecule, and any combination thereof.Reaction reagents which may modulate the stability of a closed-complexinclude, but are not limited to, non-incorporable nucleotides, incorrectnucleotides, nucleotide analogs, modified polymerases, template nucleicacids with non-extendible polymerization initiation sites, and anycombination thereof.

The examination reaction mixture can include other molecules including,but not limited to, enzymes. Optionally, the examination reactionmixture includes any reagents or biomolecules generally present in anucleic acid polymerization reaction. Reaction components may include,but are not limited to, salts, buffers, small molecules, metals, andions. Optionally, salts used in the examination reaction mixture includesalts that provide monovalent cations. Optionally, properties of thereaction mixture may be manipulated, for example, electrically,magnetically, and/or with vibration.

Nucleotides and Nucleotide Analogs

Nucleotides useful for carrying out the sequencing-by-binding proceduresdescribed herein include native nucleotides, labeled nucleotides (e.g.,nucleotides that include an exogenous fluorescent dye or other label notfound in native nucleotides), and nucleotide analogs (e.g., nucleotideshaving a reversible terminator moiety).

There is flexibility in the nature of the nucleotides that may beemployed in connection with the presently described technique. Anucleotide may include as its nitrogenous base any of: adenine,cytosine, guanine, thymine, or uracil. Optionally, a nucleotide includesinosine, xanthanine, hypoxanthanine, isocytosine, isoguanine,nitropyrrole (including 3-nitropyrrole) or nitroindole (including5-nitroindole) base. Useful nucleotides include, but are not limited to,ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP,dCTP, dGTP, dUTP, dADP, dTDP, dCDP, dGDP, dUDP, dAMP, dTMP, dCMP, dGMP,and dUMP. Optionally, the phosphate group is modified with a moiety. Themoiety may include a detectable label. Optionally, the 3′ OH group ofthe nucleotide is modified with a moiety, where the moiety may be a 3′reversible or irreversible terminator moiety. Optionally, the 2′position of the nucleotide is modified with a moiety, where the moietymay be a 2′ reversible or irreversible terminator moiety. Optionally,the base of the nucleotide is modified to include a detectable label(e.g., a detectable moiety). Optionally, the base of the nucleotide ismodified to include a reversible terminator moiety. Nucleotides may alsocontain terminating inhibitors of DNA polymerase, dideoxynucleotides or2′,3′ dideoxynucleotides, which are abbreviated as ddNTPs (ddGTP, ddATP,ddTTP, ddCTP, and ddUTP).

Optionally, a closed-complex of an examination step includes anucleotide analog or modified nucleotide to facilitate stabilization ofthe closed-complex. Optionally, a nucleotide analog includes anitrogenous base, five-carbon sugar, and phosphate group and anycomponent of the nucleotide may be modified and/or replaced. Nucleotideanalogs may be non-incorporable nucleotides. Non-incorporablenucleotides may be modified to become incorporable at any point duringthe sequencing method.

Nucleotide analogs include, but are not limited to, alpha-phosphatemodified nucleotides, alpha-beta nucleotide analogs, beta-phosphatemodified nucleotides, beta-gamma nucleotide analogs, gamma-phosphatemodified nucleotides, caged nucleotides, or ddNTPs. Examples ofnucleotide analogs are described in U.S. Pat. No. 8,071,755, which isincorporated by reference herein in its entirety.

Nucleotide analogs can include terminators that reversibly preventnucleotide incorporation to the 3′-end of the primer. One type ofreversible terminator is a 3′-O-blocked reversible terminator. Theterminator is linked to the oxygen atom of the 3′ OH end of the 5-carbonsugar of a nucleotide. Another type of reversible terminator is a3′-unblocked reversible terminator. The terminator is linked to thenitrogenous base of a nucleotide. For reviews of nucleotide analogshaving terminators, see, e.g., Mu, R., et al., “The History and Advancesof Reversible Terminators Used in New Generations of SequencingTechnology,” Genomics, Proteomics & Bioinformatics 11(1):34-40 (2013),which is incorporated by reference herein in its entirety.

Optionally, nucleotides are substituted for modified nucleotide analogshaving terminators that irreversibly prevent nucleotide incorporation tothe 3′-end of the primer. Irreversible nucleotide analogs includedideoxynucleotides, ddNTPs (ddGTP, ddATP, ddTTP, ddCTP).Dideoxynucleotides lack the 3′-OH group of dNTPs that is essential forpolymerase-mediated synthesis.

Optionally, non-incorporable nucleotides include a blocking moiety thatinhibits or prevents the nucleotide from forming a covalent linkage to asecond nucleotide (3′ OH of a primer) during the incorporation step of anucleic acid polymerization reaction. The blocking moiety can be removedfrom the nucleotide, allowing for nucleotide incorporation.

Optionally, a nucleotide analog present in a closed-complex renders theclosed-complex stable. Optionally, the nucleotide analog isnon-incorporable. Optionally, the nucleotide analog is released and anative nucleotide is incorporated. Optionally, the closed-complex isreleased, the nucleotide analog is modified, and the modified nucleotideanalog is incorporated. Optionally, the closed-complex is released underreaction conditions that modify and/or destabilize the nucleotide analogin the closed-complex.

Optionally, a nucleotide analog present in a closed-complex isincorporated and the closed-complex is stabilized. The closed-complexmay be stabilized by the nucleotide analog, or for example, by anystabilizing methods disclosed herein. Optionally, the nucleotide analogdoes not allow for the incorporation of a subsequent nucleotide. Theclosed-complex can be released, for example, by any methods describedherein, and the nucleotide analog is modified. The modified nucleotideanalog may allow for subsequent incorporation of a nucleotide to its3′-end.

Optionally, a nucleotide analog is present in the reaction mixtureduring the examination step. For example, 1, 2, 3, 4 or more nucleotideanalogs are present in the reaction mixture during the examination step.Optionally, a nucleotide analog is replaced, diluted, or sequesteredduring an incorporation step. Optionally, a nucleotide analog isreplaced with a native nucleotide. The native nucleotide may include anext correct nucleotide. Optionally, a nucleotide analog is modifiedduring an incorporation step. The modified nucleotide analog can besimilar to or the same as a native nucleotide.

Optionally, a nucleotide analog has a different binding affinity for apolymerase than a native nucleotide. Optionally, a nucleotide analog hasa different interaction with a next base than a native nucleotide.Nucleotide analogs and/or non-incorporable nucleotides may base-pairwith a complementary base of a template nucleic acid.

Optionally, a nucleotide analog is a nucleotide, modified or native,fused to a polymerase. Optionally, a plurality of nucleotide analogsincludes fusions to a plurality of polymerases, wherein each nucleotideanalog includes a different polymerase.

A nucleotide can be modified to favor the formation of a closed-complexover the formation of a binary complex. A nucleotide may be selected ormodified to have a high affinity for a polymerase, wherein thepolymerase binds to a nucleotide prior to binding to the templatenucleic acid.

Any nucleotide modification that traps the polymerase in aclosed-complex may be used in the methods disclosed herein. Thenucleotide may be trapped permanently or transiently. Optionally, thenucleotide analog is not the means by which a closed-complex isstabilized. Any closed-complex stabilization method may be combined in areaction utilizing a nucleotide analog.

Optionally, a nucleotide analog that allows for the stabilization of aclosed-complex is combined with reaction conditions that usually releasethe closed-complex. The conditions include, but are not limited to, thepresence of a release reagent (e.g., catalytic metal ion, such asmagnesium or manganese). Optionally, the closed-complex is stabilizedeven in the presence of a catalytic metal ion. Optionally, theclosed-complex is released even in the presence of a nucleotide analog.Optionally, the stabilization of the closed-complex is dependent, inpart, on the concentrations and/or identity of the stabilization reagentand/or release reagents, and any combination thereof. Optionally,stabilization of a closed-complex containing nucleotide analogs iscombined with additional reaction conditions that function to stabilizea closed-complex, including, but not limited to, sequestering, removing,reducing, omitting, and/or chelating a catalytic metal ion; the presenceof a polymerase inhibitor, cross-linking agent; and any combinationthereof.

Optionally, one or more nucleotides can be labeled with distinguishingand/or detectable tags or labels; however, such tags or labels are notdetected during examination, identification of the base or incorporationof the base, and are not detected during the sequencing methodsdisclosed herein. The tags may be distinguishable by means of theirdifferences in fluorescence, Raman spectrum, charge, mass, refractiveindex, luminescence, length, or any other measurable property. The tagmay be attached to one or more different positions on the nucleotide, solong as the fidelity of binding to the polymerase-nucleic acid complexis sufficiently maintained to enable identification of the complementarybase on the template nucleic acid correctly. Optionally, the tag isattached to the nucleobase position of the nucleotide. Under suitablereaction conditions, the tagged nucleotides may be enclosed in aclosed-complex with the polymerase and the primed template nucleic acid.Alternatively, a tag is attached to the gamma phosphate position of thenucleotide.

Optionally, the labeled nucleotide can include 3-10 or more phosphategroups. Optionally, the labeled nucleotide can be any of adenosine,guanosine, cytidine, thymidine or uridine, or any other type of labelednucleotide. Optionally, the label can be an energy transfer acceptorreporter moiety. Optionally, the label can be a fluorescent dye.Optionally, the polymerase can be contacted with more than one type oflabeled nucleotide (e.g., A, G, C, and/or T/U, or others). Optionally,each type of labeled nucleotide can be operably linked to a differentreporter moiety to permit nucleotide identification. Optionally, eachtype of labeled nucleotide can be operably linked to the same type ofreporter moiety. Optionally, the labeled nucleotides are operably linkedat the terminal phosphate group with a reporter moiety. Optionally, thelabeled nucleotides are operably linked at the base moiety with areporter moiety. Optionally, the labeled nucleotide can be anon-incorporable nucleotide. Optionally, the non-incorporable nucleotidecan bind to the polymerase and primed template nucleic acid molecule ina template-dependent manner, but does not incorporate. Optionally,different types of labeled nucleotides can be employed in the method fordetecting the presence of a transiently-bound nucleotide in order todetermine the frequency, duration, or intensity, of a transiently-boundnucleotide. For example, a comparison can be made between thefrequency/duration/intensity of transiently-bound complementary andnon-complementary nucleotides. Under circumstances involving directexcitation of the reporter moiety, the length of the transient bindingtime of a complementary nucleotide can be longer and/or more frequentcompared to that of a non-complementary nucleotide.

Polymerases

Polymerases useful for carrying out the disclosed sequencing-by-bindingtechnique include naturally occurring polymerases and modified variantsthereof, including, but not limited to, mutants, recombinants, fusions,genetic modifications, chemical modifications, synthetics, and analogs.Naturally occurring polymerases and modified variants thereof are notlimited to polymerases that retain the ability to catalyze apolymerization reaction. Optionally, the naturally occurring and/ormodified variations thereof retain the ability to catalyze apolymerization reaction. Optionally, the naturally occurring and/ormodified variations have special properties that enhance their abilityto sequence DNA, including enhanced binding affinity to nucleic acids,reduced binding affinity to nucleic acids, enhanced catalysis rates,reduced catalysis rates etc. Mutant polymerases include polymeraseswherein one or more amino acids are replaced with other amino acids(naturally or non-naturally occurring), and insertions or deletions ofone or more amino acids. Modified polymerases include polymerases thatcontain an external tag, which can be used to monitor the presence andinteractions of the polymerase. Optionally, intrinsic signals from thepolymerase can be used to monitor their presence and interactions. Thus,the provided methods can include monitoring the interaction of thepolymerase, nucleotide and template nucleic acid through detection of anintrinsic signal from the polymerase. Optionally, the intrinsic signalis a light scattering signal. For example, intrinsic signals includenative fluorescence of certain amino acids such as tryptophan, whereinchanges in intrinsic signals from the polymerase may indicate theformation of a closed-complex. Thus, in the provided methods, thepolymerase is an unlabeled polymerase and monitoring is performed in theabsence of a detectable label associated with the polymerase. Somemodified polymerases or naturally occurring polymerases, under specificreaction conditions, may incorporate only single nucleotides and mayremain bound to the primer-template after the incorporation of thesingle nucleotide. Optionally, the thumb and finger domains of thepolymerase may form transient or covalent crosslinks due to theirphysical proximity in the closed form of the polymerase. The crosslinksmay be formed, for example by native or engineered cysteines at suitablepositions on the thumb and finger domains.

The term polymerase and its variants, as used herein, also refers tofusion proteins including at least two portions linked to each other,for example, where one portion includes a peptide that can catalyze thepolymerization of nucleotides into a nucleic acid strand is linked toanother portion that includes a second moiety, such as, a reporterenzyme or a processivity-modifying domain. For example, T7 DNApolymerase includes a nucleic acid polymerizing domain and a thioredoxinbinding domain, wherein thioredoxin binding enhances the processivity ofthe polymerase. Absent the thioredoxin binding, T7 DNA polymerase is adistributive polymerase with processivity of only one to a few bases.Although DNA polymerases differ in detail, they have a similar overallshape of a hand with specific regions referred to as the fingers, thepalm, and the thumb; and a similar overall structural transition,including the movement of the thumb and/or finger domains, during thesynthesis of nucleic acids.

DNA polymerases include, but are not limited to, bacterial DNApolymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viralDNA polymerases and phage DNA polymerases. Bacterial DNA polymerasesinclude E. coli DNA polymerases I, II and III, IV and V, the Klenowfragment of E. coli DNA polymerase, Clostridium stercorarium (Cst) DNApolymerase, Clostridium thermocellum (Cth) DNA polymerase and Sulfolobussolfataricus (Sso)

DNA polymerase. Eukaryotic DNA polymerases include DNA polymerases α, β,γ, δ,

, η, ζ, λ, σ, μ, and k, as well as the Rev1 polymerase (terminaldeoxycytidyl transferase) and terminal deoxynucleotidyl transferase(TdT). Viral DNA polymerases include T4 DNA polymerase, phi-29 DNApolymerase, GA-1, phi-29-like DNA polymerases, PZA DNA polymerase,phi-15 DNA polymerase, Cp1 DNA polymerase, Cp7 DNA polymerase, T7 DNApolymerase, and T4 polymerase. Other DNA polymerases includethermostable and/or thermophilic DNA polymerases such as DNA polymerasesisolated from Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis(Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermusthermophilus (Tth) DNA polymerase, Thermus flavusu (Tfl) DNA polymerase,Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNApolymerase and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli)DNA polymerase, Pyrococcus sp. GB-D polymerase, Thermotoga maritima(Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase,Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase,Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius(Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase;Sulfolobus acidocaldarius DNA polymerase; Thermococcus sp. go N-7 DNApolymerase; Pyrodictium occultum DNA polymerase; Methanococcus voltaeDNA polymerase; Methanococcus thermoautotrophicum DNA polymerase;Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNApolymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcushorikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase;Thermococcus fumicolans DNA polymerase; Aeropyrum pernix DNA polymerase;and the heterodimeric DNA polymerase DP1/DP2. Engineered and modifiedpolymerases also are useful in connection with the disclosed techniques.For example, modified versions of the extremely thermophilic marinearchaea Thermococcus species 9° N (e.g., Therminator DNA polymerase fromNew England BioLabs Inc.; Ipswich, Mass.) can be used. Still otheruseful DNA polymerases, including the 3PDX polymerase are disclosed inU.S. Pat. No. 8,703,461, the disclosure of which is incorporated byreference in its entirety.

RNA polymerases include, but are not limited to, viral RNA polymerasessuch as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and K11polymerase; Eukaryotic RNA polymerases such as RNA polymerase I, RNApolymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymeraseV; and Archaea RNA polymerases.

Reverse transcriptases include, but are not limited to, HIV-1 reversetranscriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2reverse transcriptase from human immunodeficiency virus type 2, M-MLVreverse transcriptase from the Moloney murine leukemia virus, AMVreverse transcriptase from the avian myeloblastosis virus, andtelomerase reverse transcriptase that maintains the telomeres ofeukaryotic chromosomes.

Optionally, a polymerase is tagged with a chemiluminescent tag, whereinclosed-complex formation is monitored as a stable luminescence signal inthe presence of the appropriate luminescence triggers. The unstableinteraction of the polymerase with the template nucleic acid in thepresence of an incorrect nucleotide results in a measurably weakersignal compared to the closed-complex formed in the presence of the nextcorrect nucleotide. Additionally, a wash step prior to triggeringluminescence could remove all polymerase molecules not bound in a stableclosed-complex.

Optionally, a polymerase is tagged with an optical scattering tag,wherein closed-complex formation is monitored as a stable opticalscattering signal. The unstable interaction of the polymerase with thenucleic acid in the presence of an incorrect nucleotide results in ameasurably weaker signal compared to the closed-complex formed in thepresence of the next correct nucleotide.

Optionally, the polymerase is tagged with a plasmonic nanoparticle tag,wherein the closed-complex formation is monitored as a shift inplasmonic resonance that is different from the plasmonic resonance inthe absence of the closed-complex or the presence of a closed-complexincluding an incorrect nucleotide. The change in plasmon resonance maybe due to the change in local dielectric environment in theclosed-complex, or it may be due to the synchronous aggregation of theplasmonic nanoparticles on a cluster of clonally amplified nucleic acidmolecules or another means that affects the plasmons differently in theclosed-complex configuration.

Optionally, the polymerase is tagged with a Raman scattering tag,wherein the closed-complex formation is monitored as a stable Ramanscattering signal. The unstable interaction of polymerase with thenucleic acid in the presence of an incorrect nucleotide results in ameasurably weaker signal compared to the closed-complex formed in thepresence of the next correct nucleotide.

Optionally, a next correct nucleotide is identified by a tag on apolymerase selected or modified to have a high affinity for nucleotides,wherein the polymerase binds to a nucleotide prior to binding to thetemplate nucleic acid. For example, the DNA polymerase X from theAfrican Swine Fever virus has an altered order of substrate binding,where the polymerase first binds to a nucleotide, then binds to thetemplate nucleic acid. Optionally, a polymerase is incubated with eachtype of nucleotide in separate compartments, where each compartmentcontains a different type of nucleotide and where the polymerase islabeled differently with a tag depending on the nucleotide with which itis incubated. In these conditions, unlabeled nucleotides are bound todifferently labeled polymerases. The polymerases may be the same kind ofpolymerase bound to each nucleotide type or different polymerases boundto each nucleotide type. The differentially tagged polymerase-nucleotidecomplexes may be added simultaneously to any step of the sequencingreaction. Each polymerase-nucleotide complex binds to a template nucleicacid whose next base is complementary to the nucleotide in thepolymerase-nucleotide complex. The next correct nucleotide is identifiedby the tag on the polymerase carrying the nucleotide. The interrogationof the next template base by the labeled polymerase-nucleotide complexmay be performed under non-incorporating and/or examination conditions,where once the identity of the next template base is determined, thecomplex is destabilized and removed, sequestered, and/or diluted and aseparate incorporation step is performed in a manner ensuring that onlyone nucleotide is incorporated.

A common method of introducing a detectable tag on a polymeraseoptionally involves chemical conjugation to amines or cysteines presentin the non-active regions of the polymerase.

Such conjugation methods are well known in the art. As non-limitingexamples, n-hydroxysuccinimide esters (NHS esters) are commonly employedto label amine groups that may be found on an enzyme. Cysteines readilyreact with thiols or maleimide groups, while carboxyl groups may bereacted with amines by activating them with EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride).Optionally, N-hydroxysuccinimide (NHS) chemistry is employed at pHranges where only the N-terminal amines are reactive (for instance, pH7), such that only a single tag is added per polymerase.

Optionally, the tag attached to the polymerase is a charge tag, suchthat the formation of stable closed-complex can be detected byelectrical means by measuring changes in local charge density around thetemplate nucleic acids. Methods for detecting electrical charges arewell known in the art, including methods such as field-effecttransistors, dielectric spectroscopy, impedance measurements, and pHmeasurements, among others. Field-effect transistors include, but arenot limited to, ion-sensitive field-effect transistors (ISFET),charge-modulated field-effect transistors, insulated-gate field-effecttransistors, metal oxide semiconductor field-effect transistors andfield-effect transistors fabricated using semiconducting single wallcarbon nanotubes.

Optionally, a charge tag is a peptide tag having an isoelectric pointbelow about 4 or above about 10. Optionally, a polymerase including apeptide tag has a total isoelectric point below about 5 or above about9. A charge tag may be any moiety which is positively or negativelycharged. The charge tag may include additional moieties including massand/or labels such as dyes. Optionally, the charge tag possesses apositive or negative charge only under certain reaction conditions suchas changes in pH.

A polymerase may be labeled with a fluorophore and/or quencher.Optionally, a nucleic acid is labeled with a fluorophore and/orquencher. Optionally, one or more nucleotides are labeled with afluorophore and/or quencher. Exemplary fluorophores include, but are notlimited to, fluorescent nanocrystals; quantum dots; d-Rhodamine acceptordyes including dichloro[R110], dichloro[R6G], dichloro[TAMRA],dichloro[ROX] or the like; fluorescein donor dye including fluorescein,6-FAM, or the like; Cyanine dyes such as Cy3B; Alexa dyes, SETA dyes,Atto dyes such as atto 647N which forms a FRET pair with Cy3B and thelike. Fluorophores include, but are not limited to, MDCC(7-diethylamino-3-[([(2-maleimidyl)ethyl]amino)carbonyl]coumarin), TET,HEX, Cy3, TMR, ROX, Texas Red, Cy5, LC red 705 and LC red 640.Fluorophores and methods for their use including attachment topolymerases and other molecules are described in The Molecular Probes®Handbook (Life Technologies; Carlsbad Calif.) and Fluorophores Guide(Promega; Madison, Wis.), which are incorporated herein by reference intheir entireties. Exemplary quenchers include, but are not limited to,ZEN, IBFQ, BHQ-1, BHQ-2, DDQ-I, DDQ-11, Dabcyl, Qx1 quencher, Iowa BlackRQ, and IRDye QC-1.

Optionally, a conformationally sensitive dye may be attached close tothe active site of the polymerase without affecting the polymerizationability or fidelity of the polymerase; wherein a change in conformation,or a change in polar environment due to the formation of aclosed-complex is reflected as a change in fluorescence or absorbanceproperties of the dye. Common fluorophores such as Cy3 and fluoresceinare known to have strong solvatochromatic response to polymerase bindingand closed-complex formation, to the extent that the formation ofclosed-complex can be distinguished clearly from the binarypolymerase-nucleic acid complex. Optionally, the closed-complex can bedistinguished from binary complexes based on differences in fluorescenceor absorbance signals from a conformationally sensitive dye. Optionally,a solvatochromatic dye may be employed to monitor conformationaltransitions; wherein the change in local polar environment induced bythe conformational change can be used as the reporter signal.Solvatochromatic dyes include, but are not limited to, Reichart's dye,IR44, merocyanine dyes (e.g., merocyanine 540),4-[2-N-substituted-1,4-hydropyridin-4-ylidine)ethylidene]cyclohexa-2,5-dien-1-one,red pyrazolone dyes, azomethine dyes, indoaniline dyes, diazamerocyaninedyes, indigoid dyes, as exemplified by indigo, and others as well asmixtures thereof. Methods to introduce dyes or fluorophores to specificsites of a polymerase are well known in the art. As a non-limitingexample, a procedure for site specific labeling of a T7 DNA polymerasewith a dye is provided by Tsai et al., in “Site-Specific Labeling of T7DNA Polymerase with a Conformationally Sensitive Fluorophore and Its Usein Detecting Single-Nucleotide Polymorphisms,” Analytical Biochemistry384: 136-144 (2009), which is incorporated by reference herein in itsentirety.

Optionally, a polymerase is tagged with a fluorophore at a position thatcould sense closed-complex formation without interfering with thereaction. The polymerase may be a native or modified polymerase.Modified polymerases include those with one or more amino acidmutations, additions, and/or deletions. Optionally, one or more, but notall, cysteine amino acids are mutated to another amino acid, such asalanine. In this case, the remaining one or more cysteines are used forsite-specific conjugation to a fluorophore. Alternatively, one or moreamino acids are mutated to a reactive amino acid suitable forfluorophore conjugation, such as cysteines or amino acids includingprimary amines.

Optionally, binding between a polymerase and a template nucleic acid inthe presence of a correct nucleotide may induce a decrease influorescence, whereas binding with an incorrect nucleotide causes anincrease in fluorescence. Binding between a polymerase and a templatenucleic acid in the presence of a correct nucleotide may induce anincrease in fluorescence, whereas binding with an incorrect nucleotidecauses a decrease in fluorescence. The fluorescent signals may be usedto monitor the kinetics of a nucleotide-induced conformational changeand identify the next base in the template nucleic acid sequence.

Optionally, the polymerase/nucleic-acid interaction may be monitored byscattering signal originating from the polymerase or tags attached tothe polymerase, for instance, nanoparticle tags.

Conditions for Forming and Manipulating Closed-Complexes

As used herein, a closed-complex can be a ternary complex that includesa polymerase, primed template nucleic acid, and nucleotide. Theclosed-complex may be in a pre-chemistry conformation, wherein anucleotide is sequestered but not incorporated. The closed-complex mayalternatively be in a pre-translocation conformation, wherein anucleotide is incorporated by formation of a phosphodiester bond withthe 3′-end of the primer in the primed template nucleic acid. Theclosed-complex may be formed in the absence of catalytic metal ions ordeficient levels of catalytic metal ions, thereby physicallysequestering the next correct nucleotide within the polymerase activesite without chemical incorporation. Optionally, the sequesterednucleotide may be a non-incorporable nucleotide. The closed-complex maybe formed in the presence of catalytic metal ions, where theclosed-complex includes a nucleotide analog which is incorporated, but aPPi is not capable of release. In this instance, the closed-complex isstabilized in a pre-translocation conformation. Optionally, apre-translocation conformation is stabilized by chemically cross-linkingthe polymerase. Optionally, the closed-complex may be stabilized byexternal means. In some instances, the closed-complex may be stabilizedby allosteric binding of small molecules, or macromolecules such asantibodies or aptamers. Optionally, closed-complex may be stabilized bypyrophosphate analogs that bind close to the active site with highaffinity, preventing translocation of the polymerase.

As used herein, a stabilized closed-complex or stabilized ternarycomplex refers to a polymerase trapped at the polymerization initiationsite (3′-end of the primer) of the primed template nucleic acid by oneor a combinations of means, including but not limited to, crosslinkingthe thumb and finger domains in the closed conformation, binding of anallosteric inhibitor that prevents return of the polymerase to an openconformation, binding of pyrophosphate analogs that trap polymerase inthe pre-translocation step, absence of catalytic metal ions in theactive site of the polymerase, and addition of a metal ions such asnickel, tin and Sr²⁺ as substitutes for a catalytic metal ion. As such,the polymerase may be trapped at the polymerization initiation site evenafter the incorporation of a nucleotide. Therefore, the polymerase maybe trapped in the pre-chemistry conformation, pre-translocation step,post-translocation step or any intermediate step thereof. Thus, allowingfor sufficient examination and identification of the next correctnucleotide or base.

As described herein, a polymerase-based, sequencing-by-binding reactiongenerally involves providing a primed template nucleic acid with apolymerase and one or more types of nucleotides, wherein the nucleotidesmay or may not be complementary to the next base of the primed templatenucleic acid, and examining the interaction of the polymerase with theprimed template nucleic acid under conditions wherein either chemicalincorporation of a nucleotide into the primed template nucleic acid isdisabled or severely inhibited in the pre-chemistry conformation or oneor more complementary nucleotide incorporation occurs at the 3′-end ofthe primer. Optionally, wherein the pre-chemistry conformation isstabilized prior to nucleotide incorporation, preferably usingstabilizers, a separate incorporation step may follow the examinationstep to incorporate a single nucleotide to the 3′-end of the primer.Optionally, where a single nucleotide incorporation occurs, thepre-translocation conformation may be stabilized to facilitateexamination and/or prevent subsequent nucleotide incorporation.

As indicated above, the presently described methods for sequencing anucleic acid include an examination step. The examination step involvesbinding a polymerase to the polymerization initiation site of a primedtemplate nucleic acid in a reaction mixture including one or morenucleotides, and monitoring the interaction. Optionally, a nucleotide issequestered within the polymerase-primed template nucleic acid complexto form a closed-complex, under conditions in which incorporation of theenclosed nucleotide by the polymerase is attenuated or inhibited.Optionally a stabilizer is added to stabilize the ternary complex in thepresence of the next correct nucleotide. This closed-complex is in astabilized or polymerase-trapped pre-chemistry conformation. Aclosed-complex allows for the incorporation of the enclosed nucleotidebut does not allow for the incorporation of a subsequent nucleotide.This closed-complex is in a stabilized or trapped pre-translocationconformation. Optionally, the polymerase is trapped at thepolymerization site in its closed-complex by one or a combination ofmeans including, but not limited to, crosslinking of the polymerasedomains, crosslinking of the polymerase to the nucleic acid, allostericinhibition by small molecules, uncompetitive inhibitors, competitiveinhibitors, non-competitive inhibitors, and denaturation; wherein theformation of the trapped closed-complex provides information about theidentity of the next base on the nucleic acid template.

Optionally, a closed-complex is released from its trapped or stabilizedconformation, which may allow for nucleotide incorporation to the 3′-endof the template nucleic acid primer.

The closed-complex can be destabilized and/or released by modulating thecomposition of the reaction conditions. In addition, the closed-complexcan be destabilized by electrical, magnetic, and/or mechanical means.Mechanical means include mechanical agitation, for example, by usingultrasound agitation. Mechanical vibration destabilizes theclosed-complex and suppresses binding of the polymerase to the DNA.Thus, rather than a wash step where the examination reaction mixture isreplaced with an incorporation mixture, mechanical agitation may be usedto remove the polymerase from the template nucleic acid, enablingcycling through successive incorporation steps with a single nucleotideaddition per step.

Any combination of closed-complex stabilization or closed-complexrelease reaction conditions and/or methods may be combined. For example,a polymerase inhibitor that stabilizes a closed-complex may be presentin the examination reaction with a catalytic ion, which functions torelease the closed-complex. In the aforementioned example, theclosed-complex may be stabilized or released, depending on thepolymerase inhibitor properties and concentration, the concentration ofthe catalytic metal ion, other reagents and/or conditions of thereaction mixture, and any combination thereof.

The closed-complex can be stabilized under reaction conditions wherecovalent attachment of a nucleotide to the 3′-end of the primer in theprimed template nucleic acid is attenuated. Optionally, theclosed-complex is in a pre-chemistry conformation or ternary complex.Optionally, the closed-complex is in a pre-translocation conformation.The formation of this closed-complex can be initiated and/or stabilizedby modulating the availability of a catalytic metal ion that permitsclosed-complex release and/or chemical incorporation of a nucleotide tothe primer in the reaction mixture. Exemplary metal ions include, butare not limited to, magnesium, manganese, cobalt, and barium. Catalyticions may be any formulation, for example, being provided by salts suchas MgCl₂, Mg(CH₃CO₂)₂, and MnCl₂.

The selection and/or concentration of the catalytic metal ion may bebased on the polymerase and/or nucleotides in the sequencing reaction.For example, the HIV reverse transcriptase utilizes magnesium fornucleotide incorporation (N Kaushik, Biochemistry 35:11536-11546 (1996),and H P Patel, Biochemistry 34:5351-5363 (1995), which are incorporatedby reference herein in their entireties). The rate of closed-complexformation using magnesium versus manganese can be different depending onthe polymerase and the identity of the nucleotide. Thus, the stabilityof the closed-complex may differ depending on catalytic metal ion,polymerase, and/or nucleotide identity. Further, the concentration ofcatalytic ion necessary for closed-complex stabilization may varydepending on the catalytic metal ion, polymerase, and/or nucleotideidentity and can be readily determined using the guidance providedherein. For example, nucleotide incorporation may occur at highcatalytic ion concentrations of one metal ion but does not occur at lowconcentrations of the same metal ion, or vice versa. Therefore,modifying metal ion identity, metal ion concentration, polymeraseidentity, and/or nucleotide identity allows for controlled examinationreaction conditions.

The closed-complex may be formed and/or stabilized by sequestering,removing, reducing, omitting, and/or chelating a catalytic metal ionduring the examination step of the sequencing reaction so thatclosed-complex release and/or chemical incorporation does not occur.Chelation includes any procedure that renders the catalytic metal ionunavailable for nucleotide incorporation, including using EDTA and/orEGTA. A reduction includes diluting the concentration of a catalyticmetal ion in the reaction mixture. The reaction mixture can be dilutedor replaced with a solution including a non-catalytic ion, which permitsclosed-complex formation, but inhibits nucleotide incorporation.Non-catalytic ions include, but are not limited to, calcium, strontium,scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper,zinc, gallium, germanium, arsenic, selenium, rhodium, and strontium.Optionally, Ni²⁺ is provided in an examination reaction to facilitateclosed-complex formation. Optionally, Sr²⁺ is provided in an examinationreaction to facilitate closed-complex formation. Optionally, anon-catalytic metal ion and a catalytic metal ion are both present inthe reaction mixture, wherein one ion is present in a higher effectiveconcentration than the other. In the provided methods, a non-catalyticion such as cobalt can become catalytic (i.e., facilitate nucleotideincorporation) at high concentrations. Thus, optionally, a lowconcentration of a non-catalytic metal ion is used to facilitate ternarycomplex formation and a higher concentration of the non-catalytic metalion is used to facilitate incorporation.

Non-catalytic ions may be added to a reaction mixture under examinationconditions. The reaction may already include nucleotides. Optionally,non-catalytic ions are complexed to one or more nucleotides andcomplexed nucleotides are added to the reaction mixture. Non-catalyticions can complex to nucleotides by mixing nucleotides with non-catalyticions at elevated temperatures (about 80 ° C.). For example, a chromiumnucleotide complex may be added to a mixture to facilitateclosed-complex formation and stabilization. Optionally, a chromiumnucleotide complex is a chromium monodentate, bidentate, or tridentatecomplex. Optionally, a chromium nucleotide complex is an α-monodentate,or β-γ-bidentate nucleotide.

Optionally, a closed-complex is formed between a polymerase, primedtemplate nucleic acid, and nucleotide in reaction conditions includingSr²⁺, wherein Sr²⁺ promotes the formation of the closed-complex. Thepresence of Sr²⁺ can allow for the favorable formation of aclosed-complex including a next correct nucleotide over the formation acomplex including an incorrect nucleotide. The Sr²⁺ ion may be presentat concentrations from about 0.01 mM to about 30 mM. Optionally, Sr²⁺ ispresent as 10 mM SrCl₂. The formation of the closed-complex is monitoredunder examination conditions to identify the next base in the templatenucleic acid of the closed-complex. The affinity of the polymerase(e.g., Klenow fragment of E. coli DNA polymerase I, Bst) for each of thedNTPs (e.g., dATP, dTTP, dCTP, dGTP) in the presence of Sr²⁺ can bedifferent. Therefore, examination can involve measuring the bindingaffinities of polymerase-template nucleic acids to dNTPs; whereinbinding affinity is indicative of the next base in the template nucleicacid. Optionally, the binding interaction may be performed underconditions that destabilize the binary interactions between thepolymerase and primed template nucleic acid. Optionally, the bindinginteraction may be performed under conditions that stabilize the ternaryinteractions between the polymerase, the primed template nucleic acid,and the next correct nucleotide. After examination, a wash step removesunbound nucleotides, and Mg²⁺ is added to the reaction to inducepyrophosphate (PPi) cleavage and nucleotide incorporation. Optionally,the wash step includes Sr²⁺ to maintain the stability of the ternarycomplex, preventing the dissociation of the ternary complex. Thereaction may be repeated until a desired sequence read-length isobtained.

Optionally, a closed-complex is formed between a polymerase, primedtemplate nucleic acid, and nucleotide in reaction conditions includingNi²⁺, wherein Ni²⁺ promotes the formation of the closed-complex. Thepresence of Ni²⁺ can allow for the favorable formation of aclosed-complex including a next correct nucleotide over the formation acomplex including an incorrect nucleotide. The Ni²⁺ ion may be presentat concentrations from about 0.01 mM to about 30 mM. Optionally, Ni²⁺ ispresent as 10 mM NiCl₂. The formation of the closed-complex is monitoredunder examination conditions to identify the next base in the templatenucleic acid of the closed-complex. The affinity of the polymerase(e.g., Klenow fragment of E. coli DNA polymerase I, Bst) for each of thedNTPs (e.g., dATP, dTTP, dCTP, dGTP) in the presence of Sr²⁺ can bedifferent. Therefore, examination can involve measuring the bindingaffinities of polymerase-template nucleic acids to dNTPs; whereinbinding affinity is indicative of the next base in the template nucleicacid. Optionally, the binding interaction may be performed underconditions that destabilize the binary interactions between thepolymerase and primed template nucleic acid. Optionally, the bindinginteraction may be performed under conditions that stabilize the ternaryinteractions between the polymerase, the primed template nucleic acid,and the next correct nucleotide. After examination, a wash removesunbound nucleotides and polymerase, and Mg²⁺ is added to the reaction toinduce pyrophosphate (PPi) cleavage and nucleotide incorporation.Optionally, the wash buffer includes Ni²⁺ to maintain the stability ofthe ternary complex, preventing the dissociation of the ternary complex.The reaction may be repeated until a desired sequence read length isobtained.

Optionally, a closed-complex is formed between a polymerase, primedtemplate nucleic acid, and nucleotide in reaction conditions includingnon-catalytic concentrations of Co²⁺, wherein Co²⁺ promotes theformation of the closed-complex. The presence of non-catalyticconcentrations of Co²⁺ can allow for the favorable formation of aclosed-complex including a next correct nucleotide over the formation acomplex including an incorrect nucleotide. The Co²⁺ ion may be presentat concentrations from about 0.01 mM to about 0.5 mM. Optionally, Co²⁺is present as 0.5 mM CoCl₂. The formation of the closed-complex ismonitored under examination conditions to identify the next base in thetemplate nucleic acid of the closed-complex. The affinity of thepolymerase (e.g., Klenow fragment of E. coli DNA polymerase I, Bst) foreach of the dNTPs (e.g., dATP, dTTP, dCTP, dGTP) in the presence of Co²⁺can be different. Therefore, examination can involve measuring thebinding affinities of polymerase-template nucleic acids to dNTPs;wherein binding affinity is indicative of the next base in the templatenucleic acid. Optionally, the binding interaction may be performed underconditions that destabilize the binary interactions between thepolymerase and primed template nucleic acid. Optionally, the bindinginteraction may be performed under conditions that stabilize the ternaryinteractions between the polymerase, the primed template nucleic acid,and the next correct nucleotide. After examination, a wash removesunbound nucleotides and polymerase, and Co²⁺ at a catalyticconcentration is added to the reaction to induce pyrophosphate (PPi)cleavage and nucleotide incorporation. Optionally, the wash bufferincludes non-catalytic amounts of Co²⁺ to maintain the stability of theternary complex, preventing the dissociation of the ternary complex. Thereaction may be repeated until a desired sequence read length isobtained.

Optionally, a catalytic metal ion may facilitate the formation of aclosed-complex without subsequent nucleotide incorporation andclosed-complex release. Optionally, a concentration of 0.5, 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 μM Mg²⁺ in a reaction mixture can induceconformational change of a polymerase to form a closed-complex withoutsubsequent nucleotide incorporation, PPi and closed-complex release.Optionally, the concentration of Mg²⁺ is from about 0.5 μM to about 10μM, from about 0.5 μM to about 5 μM, from about 0.5 μM to about 4 μM,from about 0.5 μM to about 3 μM, from about μM, to about 5 μM, fromabout 1 μM to about 4 μM, and from about 1 μM to about 3 μM.

Optionally, the concentration of available catalytic metal ion in thesequencing reaction which is necessary to allow nucleotide incorporationis from about 0.001 mM to about 10 mM, from about 0.01 mM to about 5 mM,from about 0.01 mM to about 3 mM, from about 0.01 mM to about 2 mM, fromabout 0.01 mM to about 1 mM, from about 0.05 mM to about 10 mM, fromabout 0.05 mM to about 5 mM, from about 0.05 mM to about 3 mM, fromabout 0.05 to about 2 mM, or from about 0.05 mM to about 1 mM.Optionally, the concentration of catalytic metal ion is from 5 mM to 50mM. Optionally, the concentration of catalytic metal ion is from 5 mM to15 mM, or about 10 mM.

A non-catalytic ion may be added to the reaction mixture at any stageincluding before, during, or after any of the following reaction steps:providing a primed template nucleic acid, providing a polymerase,formation of a binary complex, providing a nucleotide, formation of apre-chemistry closed-complex, nucleotide incorporation, formation of apre-translocation closed-complex, and formation of a post-translocationconformation. The non-catalytic ion may be added to the reaction mixtureduring wash steps. The non-catalytic ion may be present through thereaction in the reaction mixture. For example, a catalytic ion is addedto the reaction mixture at concentrations which dilute the non-catalyticmetal ion, allowing for nucleotide incorporation.

The ability of catalytic and non-catalytic ions to modulate nucleotideincorporation may depend on conditions in the reaction mixtureincluding, but not limited to, pH, ionic strength, chelating agents,chemical cross-linking, modified polymerases, non-incorporablenucleotides, mechanical or vibration energy, and electric fields.

Optionally, the concentration of non-catalytic metal ion in thesequencing reaction necessary to allow for closed-complex formationwithout nucleotide incorporation is from about 0.1 mM to about 50 mM,from about 0.1 mM to about 40 mM, from about 0.1 mM to about 30 mM, fromabout 0.1 mM to about 20 mM, from about 0.1 mM to about 10 mM, fromabout 0.1 mM to about 5 mM, from about 0.1 to about 1 mM, from about 1mM to about 50 mM, from about 1 to about 40 mM, from about 1 mM to about30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM,from about 1 mM to about 5 mM, from about 2 mM to about 30 mM, fromabout 2 mM to about 20 mM, from about 2 mM to about 10 mM, or anyconcentration within these ranges.

A closed-complex may be formed and/or stabilized by the addition of apolymerase inhibitor to the examination reaction mixture. Inhibitormolecules phosphonoacetate (phosphonoacetic acid) and phosphonoformate(phosphonoformic acid, common name Foscarnet), Suramin, Aminoglycosides,INDOPY-1 and Tagetitoxin are non-limiting examples of uncompetitive ornoncompetitive inhibitors of polymerase activity. The binding of theinhibitor molecule, near the active site of the enzyme, traps thepolymerase in either a pre-translocation or post-translocation step ofthe nucleotide incorporation cycle, stabilizing the polymerase in itsclosed-complex conformation before or after the incorporation of anucleotide, and forcing the polymerase to be bound to the templatenucleic acid until the inhibitor molecules are not available in thereaction mixture by removal, dilution or chelation.

Thus, provided is a method for sequencing a template nucleic acidmolecule including an examination step including providing a templatenucleic acid molecule primed with a primer; contacting the primedtemplate nucleic acid molecule with a first reaction mixture including apolymerase, a polymerase inhibitor and at least one unlabeled nucleotidemolecule; monitoring the interaction of the polymerase with the primedtemplate nucleic acid molecule in the presence of the unlabelednucleotide molecule without incorporation of the nucleotide into theprimer of the primed template nucleic acid molecule; and identifying thenucleotide that is complementary to the next base of the primed templatenucleic acid molecule by the monitored interaction. The polymeraseinhibitor prevents the incorporation of the unlabeled nucleotidemolecule into the primer of the primer template nucleic acid.Optionally, the inhibitor is a non-competitive inhibitor, an allostericinhibitor, or an uncompetitive allosteric inhibitor. Optionally, thepolymerase inhibitor competes with a catalytic ion binding site in thepolymerase.

Detection Platforms: Instrumentation for Detecting the Closed-Complex

The interaction between the polymerase and the template nucleic acid inthe presence of nucleotides can be monitored with or without the use ofan exogenous label. For example, the sequencing reaction may bemonitored by detecting the change in refractive index, fluorescenceemission, charge detection, Raman scattering detection, ellipsometrydetection, pH detection, size detection, mass detection, surface plasmonresonance, guided mode resonance, nanopore optical interferometry,whispering gallery mode resonance, nanoparticle scattering, photoniccrystal, quartz crystal microbalance, bio-layer interferometry,vibrational detection, pressure detection and other label-free detectionschemes that detect the added mass or refractive index due to polymerasebinding in a closed-complex with a template nucleic acid.

Optionally, detecting a change in refractive index is accomplished byone or a combination of means, including, but not limited to, surfaceplasmon resonance sensing, localized plasmon resonance sensing,plasmon-photon coupling sensing, transmission sensing throughsub-wavelength nanoholes (enhanced optical transmission), photoniccrystal sensing, interferometry sensing, refraction sensing, guided moderesonance sensing, ring resonator sensing, or ellipsometry sensing.Optionally, nucleic acid molecules may be localized to a surface,wherein the interaction of polymerase with nucleic acids in the presenceof various nucleotides may be measured as a change in the localrefractive index.

Optionally, the template nucleic acid is tethered to or localizedappropriately on or near a surface, such that the interaction ofpolymerase and template nucleic acid in the presence of nucleotideschanges the light transmitted across or reflected from the surface. Thesurface may contain nanostructures. Optionally, the surface is capableof sustaining plasmons or plasmon resonance. Optionally, the surface isa photonic substrate, not limited to a resonant cavity, resonant ring orphotonic crystal slab. Optionally, the surface is a guided moderesonance sensor. Optionally, the nucleic acid is tethered to, orlocalized appropriately on or near a nanohole array, a nanoparticle or amicroparticle, such that the interaction of polymerase and templatenucleic acid in the presence of nucleotides changes the absorbance,scattering, reflection or resonance of the light interacting with themicroparticle or nanoparticle.

Optionally, a nanohole array on a gold surface is used as a refractiveindex sensor. The template nucleic acid may be attached to a metalsurface by standard thiol chemistry, incorporating the thiol group onone of the primers used in a PCR reaction to amplify the DNA. When thedimensions of the nanohole array are appropriately tuned to the incidentlight, binding of the polymerase to the template nucleic acid in thepresence of nucleotides can be monitored as a change in lighttransmitted across the nanoholes. For both the labeled and label-freeschemes, simple and straightforward measurement of equilibrium signalintensity may reveal the formation of a stable closed-complex.

Optionally, nucleic acid molecules are localized to a surface capable ofsustaining surface plasmons, wherein the change in refractive indexcaused by the polymerase interaction with localized nucleic acids may bemonitored through the change in the properties of the surface plasmons,wherein further, said properties of surface plasmons may include surfaceplasmon resonance. Surface plasmons, localized surface plasmons (LSP),or surface plasmon polaritons (SPP), arise from the coupling ofelectromagnetic waves to plasma oscillations of surface charges. LSPsare confined to nanoparticle surfaces, while SPPs and are confined tohigh electron density surfaces, at the interface between high electronmobility surfaces and dielectric media. Surface plasmons may propagatealong the direction of the interface, whereas they penetrate into thedielectric medium only in an evanescent fashion. Surface plasmonresonance conditions are established when the frequency of incidentelectromagnetic radiation matches the natural frequency of oscillationof the surface electrons. Changes in dielectric properties at theinterface, for instance due to binding or molecular crowding, affectsthe oscillation of surface electrons, thereby altering the surfaceplasmon resonance wavelength. Surfaces capable of surface plasmonresonance include, in a non-limiting manner, nanoparticles, clusters andaggregates of nanoparticles, continuous planar surfaces, nanostructuredsurfaces, and microstructured surfaces. Materials such as gold, silver,aluminum, high conductivity metal oxides (e.g., indium tin oxide, zincoxide, tungsten oxide) are capable of supporting surface plasmonresonance at their surfaces.

Optionally, a single nucleic acid molecule, or multiple clonal copies ofa nucleic acid, are attached to a nanoparticle, such that binding ofpolymerase to the nucleic acid causes a shift in the localized surfaceplasmon resonance (LSPR). Light incident on the nanoparticles inducesthe conduction electrons in them to oscillate collectively with aresonant frequency that depends on the nanoparticles' size, shape andcomposition. Nanoparticles of interest may assume different shapes,including spherical nanoparticles, nanorods, nanopyramids, nanodiamonds,and nanodiscs. As a result of these LSPR modes, the nanoparticles absorband scatter light so intensely that single nanoparticles are easilyobserved by eye using dark-field (optical scattering) microscopy. Forexample, a single 80-nm silver nanosphere scatters 445-nm blue lightwith a scattering cross-section of 3×10⁻² m², a million-fold greaterthan the fluorescence cross-section of a fluorescein molecule, and athousand fold greater than the cross-section of a similarly sizednanosphere filled with fluorescein to the self-quenching limit.Optionally, the nanoparticles are plasmon-resonant particles configuredas ultra-bright, nanosized optical scatters with a scattering peakanywhere in the visible spectrum. Plasmon-resonant particles areadvantageous as they do not bleach. Optionally, plasmon-resonantparticles are prepared, coated with template nucleic acids, and providedin a reaction mixture including a polymerase and one or morenucleotides, wherein a polymerase-template nucleic acid-particleinteraction is detected. One or more of the aforementioned steps may bebased on or derived from one or more methods disclosed by Schultz etal., in PNAS 97:996-1001 (2000), which is incorporated by referenceherein in its entirety.

The very large extinction coefficients at resonant wavelength enablesnoble-metal nanoparticles to serve as extremely intense labels fornear-surface interactions. Optionally, polymerase interaction withnanoparticle-localized DNA results in a shift in the resonantwavelength. The change in resonant wavelength due to binding or bindinginteractions can be measured in one of many ways. Optionally, theillumination is scanned through a range of wavelengths to identify thewavelength at which maximum scattering is observed at an imaging device.Optionally, broadband illumination is utilized in conjunction with adispersive element near the imaging device, such that the resonant peakis identified spectroscopically. Optionally, the nanoparticle system maybe illuminated at its resonant wavelength, or near its resonantwavelength, and any binding interactions may be observed as a drop inintensity of light scattered as the new resonant wavelength shifts awayfrom the illumination wavelength. Depending on the positioning of theilluminating wavelength, interactions may even appear as an increase innanoparticle scattering as the resonance peak shifts towards theillumination wavelength. Optionally, DNA-attached-nanoparticles may belocalized to a surface, or, alternatively, theDNA-attached-nanoparticles may be suspended in solution. A comprehensivereview of biosensing using nanoparticles is described by Anker et al.,in Nature Materials 7: 442-453 (2008), which is incorporated in itsentirety herein by reference.

Optionally, nano-features capable of LSPR are lithographically patternedon a planar substrate. The two-dimensional patterning of nano-featureshas advantages in multiplexing and high-throughput analysis of a largenumber of different nucleic acid molecules. Optionally, gold nanopostsare substrates for surface plasmon resonance imaging detection ofpolymerase-template nucleic acid interactions, wherein the nucleic acidsare attached to the nanoposts. Nanostructure size and period caninfluence surface plasmon resonance signal enhancement, optionally,providing a 2, 3, 4, 5, 6, 7, 8-fold or higher signal amplification whencompared to control films.

Optionally, surface plasmon resonance may be sustained in planarsurfaces. A number of commercial instruments based on the Kretschmannconfiguration (e.g., Biacore, Uppsala, Sweden) and surface plasmonresonance imaging (e.g., GWC Technologies; Madison, Wis.; or Horiba;Kyoto, Japan) are available and have well established protocols forattaching DNA to their surfaces, as single spots and in multiplexedarray patterns. In the Kretschmann configuration, a metal film,typically gold, is evaporated onto the side of a prism and incidentradiation is launched at an angle to excite the surface plasmons. Anevanescent wave penetrates through the metal film exciting plasmons onthe other side, where it may be used to monitor near-surface and surfaceinteractions near the gold film. At the resonant angle, the lightreflected from the prism-gold interface is severely attenuated. Assumingfixed wavelength illumination, binding interactions may be examined bymonitoring both the intensity of the reflected light at a fixed angleclose to the resonant angle, as well as by monitoring the changes inangle of incidence required to establish surface plasmon resonanceconditions (minimum reflectivity). When a 2D imaging device such as aCCD or CMOS camera is utilized to monitor the reflected light, theentire illumination area may be imaged with high resolution. This methodis called surface plasmon resonance imaging (SPRi). It allows highthroughput analysis of independent regions on the surfacesimultaneously. Broadband illumination may also be used, in a fixedangle configuration, wherein the wavelength that is coupled to thesurface plasmon resonance is identified spectroscopically by looking fordips in the reflected spectrum. Surface interactions are monitoredthrough shifts in the resonant wavelength.

Surface plasmon resonance is a well-established method for monitoringprotein-nucleic acid interactions, and there exist many standardprotocols both for nucleic acid attachment as well as for analyzing thedata. Illustrative references from the literature include Cho et al.,“Binding Kinetics of DNA-Protein Interaction Using Surface PlasmonResonance,” Protocol Exchange, May 22, 2013; and Brockman et al., “AMultistep Chemical Modification Procedure To Create DNA Arrays on GoldSurfaces for the Study of Protein-DNA Interactions with Surface PlasmonResonance Imaging,” Journal of the American Chemical Society 121:8044-51 (1999), both of which are incorporated by reference herein intheir entireties.

Polymerase/nucleic-acid interactions may be monitored on nanostructuredsurfaces capable of sustaining localized surface plasmons. Optionally,polymerase/nucleic-acid interactions may be monitored on nanostructuredsurfaces capable of sustaining surface plasmon polaritons.

Optionally, polymerase/nucleic-acid interactions may be monitored onnanostructured surfaces capable of sustaining localized surfaceplasmons. Optionally, polymerase/nucleic-acid interactions may bemonitored on nanostructured surfaces capable of sustaining surfaceplasmon polaritons.

Optionally, extraordinary optical transmission (EOT) through a nanoholesarray may be used to monitor nucleic-acid/polymerase interactions. Lighttransmitted across subwavelength nanoholes in plasmonic metal films ishigher than expected from classical electromagnetic theory. Thisenhanced optical transmission may be explained by considering plasmonicresonant coupling to the incident radiation, whereby at resonantwavelength, a larger than anticipated fraction of light is transmittedacross the metallic nanoholes. The enhanced optical transmission isdependent on the dimensions and pitch of the nanoholes, properties ofthe metal, as well as the dielectric properties of the medium on eitherside of the metal film bearing the nanoholes. In the context of abiosensor, the transmissivity of the metallic nanohole array depends onthe refractive index of the medium contacting the metal film, whereby,for instance, the interaction of polymerase with nucleic acid attachedto the metal surface may be monitored as a change in intensity of lighttransmitted across the nanoholes array. Instrumentation and alignmentrequirements when using the EOT/plasmonic nanohole array approach ofsurface plasmon resonance may be employed using very compact optics andimaging elements. Low power LED illumination and a CMOS or CCD cameramay suffice to implement robust EOT plasmonic sensors. An exemplarynanohole array-based surface plasmon resonance sensing device isdescribed by Escobedo et al., in “Integrated Nanohole Array SurfacePlasmon Resonance Sensing Device Using a Dual-Wavelength Source,”Journal of Micromechanics and Microengineering 21: 115001 (2011), whichis herein incorporated by reference in its entirety.

The plasmonic nanohole array may be patterned on an optically opaquelayer of gold (greater than 50 nm thickness) deposited on a glasssurface. Optionally, the plasmonic nanohole array may be patterned on anoptically thick film of aluminum or silver deposited on glass.Optionally, the nanohole array is patterned on an optically thick metallayer deposited on low refractive index plastic. Patterning plasmonicnanohole arrays on low refractive index plastics enhances thesensitivity of the device to refractive index changes by better matchingthe refractive indices on the two sides of the metal layer. Optionally,refractive index sensitivity of the nanohole array is increased byincreasing the distance between holes. Optionally, nanohole arrays arefabricated by replication, for example, by embossing, casting,imprint-lithography, or template-stripping. Optionally, nanohole arraysare fabricated by self-assembly using colloids. Optionally, nanoholearrays are fabricated by projection direct patterning, such as laserinterference lithography.

A nano-bucket configuration may be preferable to a nanoholeconfiguration. In the nanohole configuration, the bottom of thenano-feature is glass or plastic or other appropriate dielectric,whereas in the nano-bucket configuration, the bottom of the nano-featureincludes a plasmonic metal. The nano-bucket array advantageously isrelatively simple to fabricate while maintaining the transmissionsensitivity to local refractive index.

Optionally, the nanohole array plasmonic sensing is combined withlens-free holographic imaging for large area imaging in an inexpensivemanner. Optionally, a plasmonic biosensing platform includes a plasmonicchip with nanohole arrays, a light-emitting diode source configured toilluminate the chip, and a CMOS imager chip to record diffractionpatterns of the nanoholes, which is modulated by molecular bindingevents on the surface. The binding events may be the formation of aclosed-complex between a polymerase and a template nucleic acid in thepresence of a nucleotide.

The methods to functionalize surfaces (for nucleic acid attachment) forsurface plasmon resonance sensing may be directly applied to EOTnanohole arrays as both sensing schemes employ similar metal surfaces towhich nucleic acids need to be attached.

Optionally, the refractive index changes associated withpolymerase/nucleic acid interaction may be monitored on nanostructuredsurfaces that do not support plasmons. Optionally, guided mode resonancemay be used to monitor the polymerase/nucleic-acid interaction.Guided-mode resonance or waveguide-mode resonance is a phenomenonwherein the guided modes of an optical waveguide can be excited andsimultaneously extracted by the introduction of a phase-matchingelement, such as a diffraction grating or prism. Such guided modes arealso called “leaky modes,” as they do not remain guided and have beenobserved in one and two-dimensional photonic crystal slabs. Guided moderesonance may be considered a coupling of a diffracted mode to awaveguide mode of two optical structured placed adjacent or on top ofeach other. For instance, for a diffraction grating placed on top of anoptical waveguide, one of the diffracted modes may couple exactly intothe guided mode of the optical waveguide, resulting in propagation ofthat mode along the waveguide. For off-resonance conditions, no light iscoupled into the waveguide, so the structure may appear completelytransparent (if dielectric waveguides are used). At resonance, theresonant wavelength is strongly coupled into the waveguide and may becouple out of the structure depending on downstream elements from thegrating-waveguide interface. In cases where the grating coupler isextended over the entire surface of the waveguide, the light cannot beguided, as any light coupled in is coupled out at the next gratingelement. Therefore, in a grating waveguide structure, resonance isobserved as a strong reflection peak, whereas the structure istransparent to off-resonance conditions. The resonance conditions aredependent on angle, grating properties, polarization and wavelength ofincident light. For cases where the guided mode propagation is notpresent, for instance due to a grating couple to the entire surface ofthe waveguide, the resonant mode may also be called leaky-moderesonance, in light of the strong optical confinement and evanescentpropagation of radiation in a transverse direction from the waveguidelayer. Change in dielectric properties near the grating, for instancedue to binding of biomolecules affects the coupling into the waveguide,thereby altering the resonant conditions. Optionally, where nucleic acidmolecules are attached to the surface of grating waveguide structures,the polymerase/nucleic-acid interaction may be monitored as a change inwavelength of the leaky mode resonance.

A diffraction element may be used directly on a transparent substratewithout an explicit need for a waveguide element. The change inresonance conditions due to interactions near the grating nanostructuremay be monitored as resonant wavelength shifts in the reflected ortransmitted radiation.

Reflected light from a nucleic acid attached guided mode resonant sensormay be used to monitor the polymerase/nucleic-acid interaction. Abroadband illumination source may be employed for illumination, and aspectroscopic examination of reflected light could reveal changes inlocal refractive index due to polymerase binding.

Optionally, a broadband illumination may be used and the transmittedlight may be examined to identify resonant shifts due to polymeraseinteraction. A linearly polarized narrow band illumination may be used,and the transmitted light may be filtered through a cross-polarizer;wherein the transmitted light is completely attenuated due to thecrossed polarizers excepting for the leaky mode response whosepolarization is modified. This implementation converts refractive indexmonitoring to a simple transmission assay that may be monitored oninexpensive imaging systems. Published material describe the assembly ofthe optical components. See, Nazirizadeh et al., “Low-Cost Label-FreeBiosensors Using Photonic Crystals Embedded between Crossed Polarizers,”Optics Express 18: 19120-19128 (2010), which is incorporated herein byreference in its entirety.

In addition to nanostructured surfaces, plain, unstructured surfaces mayalso be used advantageously for monitoring refractive index modulations.Optionally, interferometry may be employed to monitor the interaction ofpolymerase with nucleic acid bound to an un-structured, opticallytransparent substrate. Nucleic acid molecules may be attached to the topsurface of a glass slide by any means known in the art, and the systemilluminated from the bottom surface of the glass slide. There are tworeflection surfaces in this configuration, one reflection from thebottom surface of the glass slide, and the other from the top surfacewhich has nucleic acid molecules attached to it. The two reflected wavesmay interfere with each other causing constructive or destructiveinterference based on the path length differences, with the wavereflected from the top surface modulated by the changes in dielectricconstant due to the bound nucleic acid molecules (and subsequently bythe interaction of polymerase with the bound nucleic acid molecules).With the reflection from the bottom surface unchanged, any binding tothe nucleic acid molecules may be reflected in the phase differencebetween the beams reflected from the top and bottom surfaces, which inturn affects the interference pattern that is observed. Optionally,bio-layer interferometry is used to monitor the nucleic acid/polymeraseinteraction. Bio-layer interferometry may be performed on commercialdevices such as those sold by Pall Forte Bio corporation (Menlo Park,Calif.).

Optionally, the reflected light from the top surface is selectivelychosen by using focusing optics. The reflected light from the bottomsurface is disregarded because it is not in the focal plane. Focusingonly on the nucleic-acid-attached top surface, the light collected bythe focusing lens includes a planar wave, corresponding to the partiallyreflected incident radiation, and a scattered wave, corresponding to theradiations scattered in the collection direction by molecules in thefocal plane. These two components may be made to interfere if theincident radiation is coherent. This scattering based interferometricdetection is extremely sensitive and can be used to detect down tosingle protein molecules.

Optionally, a field-effect transistor (FET) is configured as a biosensorfor the detection of a closed-complex. A gate terminal of the FET ismodified by the addition of template nucleic acids. The binding of apolymerase including a charged tag results in changes in electrochemicalsignals. Binding of a polymerase with a next correct nucleotide to thetemplate nucleic acid provides different signals than polymerase bindingto a template nucleic acid in the presence of other incorrectnucleotides, where each incorrect nucleotide may also provide adifferent signal. Optionally, polymerase interactions with a templatenucleic acid are monitored using FET without the use of a an exogenouslabel on the polymerase, primed template nucleic acid, or nucleotide.Optionally, the pH change that occurs due to release of H⁺ ions duringthe incorporation reaction is detected using a FET. Optionally, thepolymerase includes a tag that generates continuous H⁺ ions that isdetected by the FET. Optionally, the continuous H⁺ ion generating tag isan ATP synthase. Optionally, the continuous H⁺ ion generation tag ispalladium, copper or another catalyst. Optionally, the release of a PPiafter nucleotide incorporation is detected using FET. For example, onetype of nucleotide may be provided to a reaction at a time. Once thenext correct nucleotide is added and conditions allow for incorporation,PPi is cleaved, released, and detected using FET, therefore identifyingthe next correct nucleotide and the next base. Optionally, templatenucleic acids are bound to walls of a nanotube. Optionally, a polymeraseis bound to a wall of a nanotube. FET is advantageous for use as asequencing sensor due to its small size and low weight, making itappropriate for use as a portable sequencing monitoring component.Details of FET detection of molecular interactions are described by Kimet al., in “An FET-Type Charge Sensor for Highly Sensitive Detection ofDNA Sequence,” Biosensors and Bioelectronics, Microsensors andMicrosystems 20: 69-74 (2004), doi:10.1016/j.bios.2004.01.025; and byStar et al., in “Electronic Detection of Specific Protein Binding UsingNanotube FET Devices,” Nano Letters 3: 459-63 (2003),doi:10.1021/n10340172, which are incorporated by reference herein intheir entireties.

By way of example, the polymerase includes a fluorescent tag. To monitorpolymerase-nucleic acid interaction with high signal-to-noise,evanescent illumination or confocal imaging may be employed. Theformation of a closed-complex on localized template nucleic acids may beobserved as an increased fluorescence compared to the background, forinstance, whereas in some instances it may be also be observed as adecreased fluorescence due to quenching or change in local polarenvironment. Optionally, a fraction of polymerase molecules may betagged with a fluorophore while another fraction may be tagged with aquencher in the same reaction mixture; wherein, the formation ofclosed-complex on a localized, clonal population of nucleic acid isrevealed as decrease in fluorescence compared to the background. Theclonal population of nucleic acids may be attached to a support surfacesuch as a planar substrate, microparticle, or nanoparticle. Optionally,a polymerase is tagged with a fluorophore, luminophore,chemiluminophore, chromophore, or bioluminophore.

Optionally, a plurality of template nucleic acids is tethered to asurface and one (or more) dNTPs are flowed in sequentially. The spectrumof affinities reveals the identity of the next correct nucleotide andtherefore the next base in the template nucleic acid. Optionally, theaffinities are measured without needing to remove and replace reactionmixture conditions (i.e., a wash step). Autocorrelation of the measuredintensities of the binding interaction, for instance, could readilyreveal the dynamics of nucleic acid sequence. Optionally, examinationincludes monitoring the affinity of the polymerase to the primedtemplate nucleic acid in the presence of nucleotides. Optionally, thepolymerase binds transiently with the nucleic acid and the bindingkinetics and affinity provides information about the identity of thenext base on the template nucleic acid. Optionally, a closed-complex isformed, wherein the reaction conditions involved in the formation of theclosed-complex provide information about the next base on the nucleicacid.

Any technique that can measure dynamic interactions between a polymeraseand nucleic acid may be used to measure the affinities and enable thesequencing reaction methods disclosed herein.

Systems for Detecting Nucleotide-Specific Ternary Complex Formation

The provided methods can be performed using a platform, where anycomponent of the nucleic acid polymerization reaction is localized to asurface. Optionally, the template nucleic acid is attached to a planarsubstrate, a nanohole array, a microparticle, or a nanoparticle.Optionally, all reaction components are freely suspended in the reactionmixture, and not immobilized to a solid support substrate.

Optionally, the template nucleic acid is immobilized to a surface. Thesurface may be a planar substrate, a hydrogel, a nanohole array, amicroparticle, or a nanoparticle. Optionally, the reaction mixturescontain a plurality of clonally amplified template nucleic acidmolecules. Optionally, the reaction mixtures contain a plurality ofdistinguishable template nucleic acids.

Provided herein, inter alia, are systems for performing sequencingreactions involving the examination of the interaction between apolymerase and a primed template nucleic acid in the presence ofnucleotides to identify the next base in the template closed-complex byone or a combination of means including, but not limited to,crosslinking of the polymerase domains, crosslinking of the polymeraseto the nucleic acid, allosteric inhibition by small molecules,uncompetitive inhibitors, competitive inhibitors, non-competitiveinhibitors, and denaturation; wherein the formation of the trappedpolymerase complex provides information about the identity of the nextbase on the nucleic acid template.

Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to perform a polymerase and templatenucleic acid binding assay in the presence of nucleotides, wherein thetemplate nucleic acid is provided on a nanostructure. Optionally, thesystem includes one or more reagents and instructions necessary to bindtemplate DNA molecules onto a nanostructure. For example, the systemprovides a nanostructure, such as a chip, configured for use withsurface plasmon resonance to determine binding kinetics. An example ofsuch a chip is a CMS Sensor S chip (GE Healthcare; Piscatawany, N.J.).The system may provide instrumentation such as a surface plasmonresonance instrument. The system may provide streptavidin and/or biotin.Optionally, the system provides biotin-DNA, DNA ligase, buffers, and/orDNA polymerase for preparation of biotinylated template DNA. Optionally,the system provides a gel or reagents (e.g., phenol:chloroform) forbiotinylated DNA purification. Alternatively, the system providesreagents for biotinylated template DNA characterization, for example,mass spectrometry or HPLC. Optionally, the system includes streptavidin,a chip, reagents, instrumentation, and/or instructions forimmobilization of streptavidin on a chip. Optionally, a chip is providedin the system already configured for template DNA coating, wherein thechip is immobilized with a reagent capable of binding template nucleicacids or modified template nucleic acids (e.g., biotinylated templateDNA). Optionally, the system provides reagents for chip regeneration.

Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to perform a polymerase and templatenucleic acid binding assay in the presence of nucleotides, wherein thetemplate nucleic acid is provided on a nanoparticle. Optionally, thesystem includes one or more reagents and instructions necessary to bindtemplate DNA molecules onto a nanoparticle. The nanoparticle may beconfigured for the electrochemical detection of nucleic acid-polymeraseinteraction, for instance, by using gold nanoparticles. Optionally, theDNA-nanoparticle conjugates are formed between aqueous gold colloidsolutions and template DNA molecules including, for example, free thiolor disulfide groups at their ends. The conjugates may include samenucleic acid sequence. Optionally, the nanoparticle conjugates arestabilized against flocculation and precipitation at high temperature(e.g., greater than 60° C.) and high ionic strength (e.g., 1M Na⁺).Optionally, the system provides reagents for preparing template DNAmolecules for nanoparticle attachment, including, generating templateDNA molecules with disulfides or thiols. Disulfide-containing templatenucleic acids may be synthesized using, for example, a 3′-thiol modifiercontrolled-pore glass (CPG) or by beginning with a universal support CPGand adding a disulfide modifier phosphoramidite as the first monomer inthe sequence. The system may provide nucleic acid synthesis reagentsand/or instructions for obtaining disulfide-modified template nucleicacids. Thiol-containing template nucleic acids may also be generatedduring nucleic acid synthesis with a 5′-tritylthiol modifierphosphoramidite. The system may provide reagents and/or instructions fornanoparticle conjugate purification using for example, electrophoresisor centrifugation. Optionally, nanoparticle conjugates are used tomonitor polymerase-template nucleic acid interactions colorimetrically.In this instance, the melting temperature of the nanoparticle conjugateincreases in the presence of strong polymerase binding. Therefore, thestrength of DNA binding can be determined by the change in this meltingtransition, which is observable by a color change. The systemsoptionally include reagents and equipment for detection of the meltingtransition.

Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to perform a polymerase and templatenucleic acid binding assay in the presence of nucleotides, using adetectable polymerase. Optionally, the polymerase is detectably labeled.Optionally, the polymerase is detected using intrinsic properties of thepolymerase, for example, aromatic amino acids. Optionally, thepolymerase and template nucleic acids present in the system areconfigured for use in solution, without conjugation to a support. Thedetectable label on the polymerase may be a fluorophore, whereinfluorescence is used to monitor polymerase-template nucleic acid bindingevents. Optionally, the detectable polymerase may be used in combinationwith template nucleic acids in solution, or template nucleic acidsconjugated to a support structure. Optionally, one or more cysteineresidues of the polymerase is labeled with Cy3-maleimide. Optionally,the system includes reagents and/or instructions necessary to preparefluorescently labeled polymerase molecules. The system may includereagents and/or instructions for purification of fluorescently labeledpolymerases.

Procedural Features of the Methods

Following the examination step, where the identity of the next base hasbeen identified via formation of a closed-complex, the reactionconditions may be reset, recharged, or modified as appropriate, inpreparation for the optional incorporation step or an additionalexamination step. Optionally, the identity of the next base has beenidentified without chemically incorporating a nucleotide. Optionally,the identity of the next base is identified with chemical incorporationof a nucleotide, wherein a subsequent nucleotide incorporation has beeninhibited. Optionally, all components of the examination step, excludingthe template nucleic acid being sequenced, are removed or washed away,returning the system to the pre-examination condition. Optionally,partial components of the examination step are removed. Optionally,additional components are added to the examination step.

Optionally, reversible terminator nucleotides are used in theincorporation step to ensure one, and only one nucleotide isincorporated per cycle. No labels are required on the reversibleterminator nucleotides as the base identity is known from theexamination step. Non-fluorescently labeled reversible terminators arereadily available from commercial suppliers. Non-labeled reversibleterminator nucleotides are expected to have much faster incorporationkinetics compared to labeled reversible terminators due to their smallersteric footprint, and similar size to natural nucleotides.

Disclosed herein, in part, are reagent cycling sequencing methods,wherein sequencing reagents are introduced, one after another, for everycycle of examination and/or incorporation. Optionally, the sequencingreaction mixture includes a polymerase, a primed template nucleic acid,and at least one type of nucleotide. Optionally, the nucleotide and/orpolymerase are introduced cyclically to the sequencing reaction mixture.Optionally, the sequencing reaction mixture includes a plurality ofpolymerases, primed template nucleic acids, and nucleotides. Optionally,a plurality of nucleotides and/or a plurality of polymerases areintroduced cyclically to the sequencing reaction mixture. Optionally,the examination step of the sequencing reaction has a differentcomposition than the incorporation step of the sequencing reaction.

Optionally, one or more nucleotides are sequentially added to andremoved from the sequencing reaction. Optionally, 1, 2, 3, 4, or moretypes of nucleotides are added to and removed from the reaction mixture.For example, one type of nucleotide is added to the sequencing reaction,removed, and replaced by another type of nucleotide. Optionally, anucleotide type present during the examination step is different from anucleotide type present during the incorporation step. Optionally, anucleotide type present during one examination step is different from anucleotide type present during a sequential examination step (i.e., thesequential examination step is performed prior to an incorporationstep). Optionally, 1, 2, 3, 4 or more types of nucleotides are presentin the examination reaction mixture and 1, 2, 3, 4, or more types ofnucleotides are present in the incorporation reaction mixture.

Optionally, a polymerase is cyclically added to and removed from thesequencing reaction. One or more different types of polymerases may becyclically added to and removed from the sequencing reaction.Optionally, a polymerase type present during the examination step isdifferent from a polymerase type present during the incorporation step.A polymerase type present during one examination step may be differentfrom a polymerase type present during a sequential examination step(i.e., the sequential examination step is performed prior to anincorporation step).

Optionally, conditions such as the presence of reagents, pH,temperature, and ionic strength are varied throughout the sequencingreaction. Optionally, a metal is cyclically added to and removed fromthe sequencing reaction. For example, a catalytic metal ion may beabsent during an examination step and present during an incorporationstep. Alternatively, a polymerase inhibitor may be present during anexamination step and absent during an incorporation step. Optionally,reaction components that are consumed during the sequencing reaction aresupplemented with the addition of new components at any point during thesequencing reaction.

Nucleotides can be added one type at a time, with the polymerase, to areaction condition that favors closed-complex formation. The polymerasebinds only to the template nucleic acid if the next correct nucleotideis present. A wash step after every nucleotide addition ensures allexcess polymerases and nucleotides not involved in a closed-complex areremoved from the reaction mixture. If the nucleotides are added one at atime, in a known order, the next base on the template nucleic acid isdetermined by the formation of a closed-complex when the addednucleotide is the next correct nucleotide. The closed-complex may beidentified by both the conformational change and the increased stabilityof the polymerase-template nucleic acid-nucleotide interaction.Optionally, the stability of the closed-complex formed in the presenceof the next correct nucleotide is at least an order of magnitude greaterthan the unstable interactions of the polymerase with the templatenucleic acid in the presence of incorrect nucleotides. The use of a washstep ensures that there are no unbound nucleotides and polymerases andthat the only nucleotides present in the reaction are those sequesteredin a closed-complex with a polymerase and a template nucleic acid. Oncethe next base on the template nucleic acid is determined, the nextcorrect nucleotide sequestered in the closed-complex may be incorporatedby flowing in reaction conditions that favor dissociation ordestabilization of the closed-complex and extending the template nucleicacid primer strand by one base (incorporation). Therefore, the wash stepensures that the only nucleotide incorporated is the next correctnucleotide from the closed-complex. This reagent cycling method may berepeated and the nucleic acid sequence determined. This reagent cyclingmethod may be applied to a single template nucleic acid molecule, or tocollections of clonal populations such as PCR products or rolling-circleamplified DNA. Many different templates can be sequenced in parallel ifthey are arrayed, for instance, on a solid support. Optionally, the washstep destabilizes binary complex formation. Optionally, the washing isperformed for a duration of time that ensures that the binary complex isremoved, leaving the stabilized closed-complex in the reaction mixture.Optionally, the wash step includes washing the reaction with a highionic strength or a high pH solution.

Optionally, the incorporation step is a three stage process. In thefirst stage, all four nucleotide types are introduced into a reactionincluding a primed template nucleic acid, with a high fidelitypolymerase, in reaction conditions which favor the formation of aclosed-complex, and the next correct nucleotides are allowed to formstable closed-complexes with the template nucleic acid. In a secondstage, excess nucleotides and unbound polymerase are washed away. In athird stage, reaction conditions are modified so that the closed-complexis destabilized and the sequestered nucleotides within theclosed-complex become incorporated into the 3′-end of the templatenucleic acid primer. In an alternative approach, the second stage ismodified to remove completely any of the high-fidelity polymerase andcognate nucleotide that may have been present in the closed-complex, andthe removed components are then replaced with a second polymerase andone or more nucleotides (e.g., reversible terminator nucleotides).Formation of tight polymerase-nucleic acid complexes in theincorporation step can be enabled by standard techniques such as fusinga non-specific DNA binding domain to the polymerase (e.g., the Phusionpolymerase, which is available from Thermo Fisher Scientific; Waltham,Mass.), and utilizing high concentrations of nucleotides to ensurecorrect nucleotides are always present in the closed-complex.

Polymerase molecules bind to primed template nucleic acid molecules in afingers-closed conformation in the presence of the next correctnucleotide even in the absence of divalent metal ions that are typicallyrequired for polymerase synthesis reactions. The conformational changetraps the nucleotide complementary to the next template base within theactive site of the polymerase. Optionally, the formation of theclosed-complex may be used to determine the identity of next base on thetemplate nucleic acid. Optionally, the primed template nucleic acids maybe contacted serially by different nucleotides in the presence ofpolymerase, in the absence of catalytic divalent metal ions; wherein theformation of a closed-complex indicates the nucleotide currently incontact with the template nucleic acid is the complementary nucleotideto the next base on the nucleic acid. A known order of nucleotides (inthe presence of polymerase and absence of catalytic metal ions) broughtinto contact with the template nucleic acid ensures facileidentification of the complementary nucleotide based on the particularposition in the order that induces closed-complex formation. Optionally,an appropriate wash step may be performed after every nucleotideaddition to ensure removal of all excess enzymes and nucleotides,leaving behind only the polymerase that is bound to nucleic acids in aclosed-complex with the next correct nucleotide at the active site. Theclosed-complex may be identified by means that reveal the conformationalchange of the polymerase in the closed conformation or by means thatreveal the increased stability of thepolymerase/nucleic-acid/next-correct-nucleotide complex compared tobinary polymerase-nucleic acid complexes or compared to unstableinteractions between the polymerase, primed template nucleic acid andincorrect nucleotides.

Optionally, the process of identifying the next complementary nucleotide(examination step) includes the steps of contacting immobilized primedtemplate nucleic acids with an examination mixture including polymeraseand nucleotides of one kind under conditions that inhibit the chemicalincorporation of the nucleotide, removing unbound reagents by a washstep, detecting the presence or absence of polymerase closed-complex onthe immobilized nucleic acids, and repeating these steps serially, withnucleotides of different kinds until a closed-complex formation isdetected. The closed-complex may be identified by both theconformational change and the increased stability of thepolymerase/nucleic-acid/next-correct-nucleotide complex. The wash stepbetween successive nucleotide additions may be eliminated by the use ofdetection mechanisms that can detect the formation of the closed-complexwith high fidelity, for instance, evanescent wave sensing methods ormethods that selectively monitor signals from the closed-complex. Theexamination steps noted above may be followed by an incorporation stepincluding, contacting the closed-complex with catalytic metal ions tocovalently add the nucleotide sequestered in the closed-complex to the3′-end of the primer. Optionally, the incorporation step may include,contacting the immobilized nucleic acids with a pre-incorporationmixture including a combination of multiple types of nucleotides andpolymerase under conditions that inhibit the chemical incorporation ofthe nucleotides; wherein the pre-incorporation mixture may containadditives and solution conditions to ensure highly efficientclosed-complex formation (e.g., low-salt conditions). The methods mayalso include performing a wash step to remove unbound reagents andproviding the immobilized complexes with an incorporation mixture,including catalytic metal ions, to chemically incorporate nucleotidessequestered within the active site of the polymerase. Thepre-incorporation mixture ensures highly efficient closed-complexformation, while the wash step and incorporation mixture ensure theaddition of a single nucleotide to the 3′-end of the primer. Optionally,the incorporation step may occur directly after examination an additionof one type of nucleotide. For instance, a repeated pattern used forsequencing may include the following flow pattern (i) dATP+/polymerase,(ii) Wash, (iii) Mg²⁺, (iv) Wash, (v) dTTP+/polymerase, (vi) Wash, (vii)Mg²⁺, (viii) Wash, (ix) dCTP+/polymerase, (x) Wash (xi) Mg²⁺, (xii)Wash, (xiii) dGTP+/polymerase, (xiv) Wash, (xv) Mg²⁺, (xvi)Wash.Optionally, the repeated pattern used for sequencing may include (i)dATP+/polymerase, (ii) Wash, (iii) dTTP+/polymerase, (iv) Wash, (v)dGTP+/polymerase, (vi) Wash, (vii) dCTP+/polymerase, (viii) Wash, (ix)Pre-incorporation mixture, (x) Wash, (xi) Mg, (xii)Wash. The wash stepstypically contain metal ion chelators and other small molecules toprevent accidental incorporations during the examination steps. Afterthe incorporation step, the primer strand is typically extended by onebase. Repeating this process, sequential nucleobases of a nucleic acidmay be identified, effectively determining the nucleic acid sequence.Optionally, the examination step is performed at high salt conditions,for example, under conditions of 50 mM to 1,500 mM salt (e.g., a saltproviding monovalent cations).

For sequencing applications, it can be advantageous to minimize oreliminate fluidics and reagents exchange. Removing pumps, valves andreagent containers can allow for simplified manufacturing of smallerdevices. Disclosed herein, in part, are “all-in” sequencing methods,wherein the need to introduce reagents one after another, for everycycle of examination and/or incorporation, is eliminated. Reagents areadded only once to the reaction, and sequencing-by-synthesis isperformed by manipulating reagents already enclosed within thesequencing reaction. A scheme such as this requires a method todistinguish different nucleotides, a method to synchronize incorporationof nucleotides across a clonal population of nucleic acids and/or acrossdifferent nucleic acid molecules, and a method to ensure only onenucleotide is added per cycle.

Optionally, the sequencing reaction mixture includes a polymerase, aprimed template nucleic acid, and at least one type of nucleotide.Optionally, the sequencing reaction mixture includes a plurality ofpolymerases, primed template nucleic acids, and nucleotides. As providedherein, a polymerase refers to a single polymerase or a plurality ofpolymerases. As provided herein, a primed template nucleic acid ortemplate nucleic acid refers to a single primed template nucleic acid orsingle template nucleic acid, or a plurality of primed template nucleicacids or a plurality of template nucleic acids. As provided herein, anucleotide refers to one nucleotide or a plurality of nucleotides. Asprovided herein, a single nucleotide is one nucleotide. Optionally, thesequencing reaction nucleotides include, but are not limited to, 1, 2,3, or 4 of the following nucleotides: dATP, dGTP, dCTP, dTTP, and dUTP.

Optionally, the examination step and the incorporation step take placein a single sequencing reaction mixture.

Optionally, 1, 2, 3, 4 or more types of nucleotides (e.g., dATP, dGTP,dCTP, dTTP) are present in the reaction mixture together at the sametime, wherein one type of nucleotide is a next correct nucleotide. Thereaction mixture further includes at least one polymerase and at leastone primed template nucleic acids. Optionally, the template nucleic acidis a clonal population of template nucleic acids. Optionally, thepolymerase, primed template nucleic acid, and the nucleotide form aclosed-complex under examination reaction conditions.

In the provided methods, four types of nucleotides can be present atdistinct and different concentrations wherein the diffusion and bindingtimes of the polymerase to the template nucleic acid are different foreach of the four nucleotides, should they be the next correctnucleotide, due to the different concentrations of the four nucleotides.For example, the nucleotide at the highest concentration would bind toits complementary base on the template nucleic acid at a fast time, andthe nucleotide at the lowest concentration would bind to itscomplementary base on the template nucleic acid at a slower time;wherein binding to the complementary base on the template nucleic acidrefers to the polymerase binding to the template nucleic acid with thenext correct nucleotide in a closed closed-complex. The identity of thenext correct nucleotide is therefore determined by monitoring the rateor time of binding of polymerase to the template nucleic acid in aclosed-complex. Optionally, the four types of nucleotides may bedistinguished by their concentration, wherein the differentconcentrations of the nucleotides result in measurably differenton-rates for the polymerase binding to the nucleic acid. Optionally, thefour types of nucleotides may be distinguished by their concentration,wherein the different concentrations of the nucleotides result inmeasurably different on-rates for the formation of a stabilizedclosed-complex.

Optionally, the polymerase is labeled. In some instances, the polymeraseis not labeled (i.e., does not harbor an exogenous label, such as afluorescent label) and any label-free detection method disclosed hereinor known in the art is employed. Optionally, the binding of thepolymerase to the nucleic acid is monitored via a detectable feature ofthe polymerase. Optionally, the formation of a stabilized closed-complexis monitored via a detectable feature of the polymerase. A detectablefeature of the polymerase may include, but is not limited to, optical,electrical, thermal, colorimetric, mass, and any combination thereof

Optionally, 1, 2, 3, 4, or more nucleotides types (e.g., dATP, dTTP,dCTP, dGTP) are tethered to 1, 2, 3, 4, or more different polymerases;wherein each nucleotide type is tethered to a different polymerase andeach polymerase has a different exogenous label or a detectable featurefrom the other polymerases to enable its identification. All tetherednucleotide types can be added together to a sequencing reaction mixtureforming a closed-complex including a tethered nucleotide-polymerase; theclosed-complex is monitored to identify the polymerase, therebyidentifying the next correct nucleotide to which the polymerase istethered. The tethering may occur at the gamma phosphate of thenucleotide through a multi-phosphate group and a linker molecule. Suchgamma-phosphate linking methods are standard in the art, where afluorophore is attached to the gamma phosphate linker. Optionally,different nucleotide types are identified by distinguishable exogenouslabels. Optionally, the distinguishable exogenous labels are attached tothe gamma phosphate position of each nucleotide.

Optionally, the sequencing reaction mixture includes a catalytic metalion. Optionally, the catalytic metal ion is available to react with apolymerase at any point in the sequencing reaction in a transientmanner. To ensure robust sequencing, the catalytic metal ion isavailable for a brief period of time, allowing for a single nucleotidecomplementary to the next base in the template nucleic acid to beincorporated into the 3′-end of the primer during an incorporation step.In this instance, no other nucleotides, for example, the nucleotidescomplementary to the bases downstream of the next base in the templatenucleic acid, are incorporated. Optionally, the catalytic metal ionmagnesium is present as a photocaged complex (e.g., DM-Nitrophen) in thesequencing reaction mixture such that localized UV illumination releasesthe magnesium, making it available to the polymerase for nucleotideincorporation. Furthermore, the sequencing reaction mixture may containEDTA, wherein the magnesium is released from the polymerase active siteafter catalytic nucleotide incorporation and captured by the EDTA in thesequencing reaction mixture, thereby rendering magnesium incapable ofcatalyzing a subsequent nucleotide incorporation.

Thus, in the provided methods, a catalytic metal ion can be present in asequencing reaction in a chelated or caged form from which it can bereleased by a trigger. For example, the catalytic metal ion catalyzesthe incorporation of the closed-complex next correct nucleotide, and, asthe catalytic metal ion is released from the active site, it issequestered by a second chelating or caging agent, disabling the metalion from catalyzing a subsequent incorporation. The localized release ofthe catalytic metal ion from its cheating or caged complex is ensured byusing a localized uncaging or un-chelating scheme, such as an evanescentwave illumination or a structured illumination. Controlled release ofthe catalytic metal ions may occur for example, by thermal means.Controlled release of the catalytic metal ions from their photocagedcomplex may be released locally near the template nucleic acid byconfined optical fields, for instance by evanescent illumination such aswaveguides or total internal reflection microscopy. Controlled releaseof the catalytic metal ions may occur for example, by altering the pH ofthe solution near the vicinity of the template nucleic acid. Chelatingagents such as EDTA and EGTA are pH dependent. At a pH below 5, divalentcations Mg²⁺ and Mn²⁺ are not effectively chelated by EDTA. A method tocontrollably manipulate the pH near the template nucleic acid allows thecontrolled release of a catalytic metal ion from a chelating agent.Optionally, the local pH change is induced by applying a voltage to thesurface to which the nucleic acid is attached. The pH method offers anadvantage in that metal goes back to its chelated form when the pH isreverted back to the chelating range.

Optionally, a catalytic metal ion is strongly bound to the active siteof the polymerase, making it necessary to remove the polymerase from thetemplate nucleic acid after a single nucleotide incorporation. Theremoval of polymerase may be accomplished by the use of a highlydistributive polymerase, which falls off the 3′-end of the strand beingsynthesized (e.g., primer) after the addition of every nucleotide. Theunbound polymerase may further be subjected to an electric or magneticfield to remove it from the vicinity of the nucleic acid molecules. Anymetal ions bound to the polymerase may be sequestered by chelatingagents present in the sequencing reaction mixture, or by molecules whichcompete with the metal ions for binding to the active site of thepolymerase without disturbing the formation of the closed-complex. Theforces which remove or move the polymerase away from the templatenucleic acid (e.g., electric field, magnetic field, and/or chelatingagent) may be terminated, allowing for the polymerase to approach thetemplate nucleic acid for another round of sequencing (i.e., examinationand incorporation). The next round of sequencing, in a non-limitingexample, includes the formation of a closed-complex, removing unboundpolymerase away from the vicinity of the template nucleic acid and/orclosed-complex, controlling the release of a catalytic metal ion toincorporate a single nucleotide sequestered within the closed-complex,removing the polymerase which dissociates from the template nucleic acidafter single incorporation away from the vicinity of the templatenucleic acid, sequestering any free catalytic metal ions through the useof chelating agents or competitive binders, and allowing the polymeraseto approach the template nucleic acid to perform the next cycle ofsequencing.

Described above are polymerase-nucleic acid binding reactions for theidentification of a nucleic acid sequence. However, nucleic acidsequence identification may include information regarding nucleic acidmodifications, including methylation and hydroxymethylation. Methylationmay occur on cytosine bases of a template nucleic acid. DNA methylationmay stably alter the expression of genes. DNA methylation is alsoindicated in the development of various types of cancers,atherosclerosis, and aging. DNA methylation therefore can serve as anepigenetic biomarker for human disease.

Optionally, one or more cytosine methylations on a template nucleic acidare identified during the sequencing by binding methods provided herein.The template nucleic acid may be clonally amplified prior to sequencing,wherein the amplicons include the same methylation as their templatenucleic acid. Amplification of the template nucleic acids may includethe use of DNA methyltransferases to achieve amplicon methylation. Thetemplate nucleic acids or amplified template nucleic acids are providedto a reaction mixture including a polymerase and one or more nucleotidetypes, wherein the interaction between the polymerase and nucleic acidsis monitored. Optionally, the interaction between the polymerase andtemplate nucleic acid in the presence of a methylated cytosine isdifferent than the interaction in the presence of an unmodifiedcytosine. Therefore, based on examination of a polymerase-nucleic acidinteraction, the identity of a modified nucleotide is determined.

Optionally, following one or more examination and/or incorporationsteps, a subset of nucleotides is added to reduce or reset phasing.Thus, the methods can include one or more steps of contacting a templatenucleic acid molecule being sequenced with a composition comprising asubset of nucleotides and an enzyme for incorporating the nucleotidesinto the strand opposite the template strand of the nucleic acidmolecule. The contacting can occur under conditions to reduce phasing inthe nucleic acid molecule. Optionally, the step of contacting thetemplate nucleic acid molecule occurs after an incorporation step and/orafter an examination step. Optionally, the contacting occurs after 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75,80, 85, 90, 95, or 100 rounds or more of sequencing, i.e., rounds ofexamination and incorporation. Optionally, the contacting occurs after30 to 60 rounds of sequencing. Optionally, the contacting occurs afterevery round of sequencing (i.e., after one set of examination andincorporation steps). Optionally, multiple contacting steps occur afterevery round of sequencing, wherein each contacting step may comprisedifferent subsets of nucleotides. Optionally, the method furthercomprises one or more washing steps after contacting. Optionally, thesubset comprises two or three nucleotides. Optionally, the subsetcomprises three nucleotides. Optionally, the subset of nucleotides isselected from three of dATP, dGTP, dCTP, dTTP or a derivative thereof.Optionally, the three nucleotides comprise adenosine, cytosine, andguanine. Optionally, the three nucleotides comprise adenosine, cytosine,and thymine. Optionally, the three nucleotides comprise cytosine,guanine and thymine. Optionally, the three nucleotides compriseadenosine, guanine and thymine. Optionally, each round of contactingcomprises the same subset or different subsets of nucleotides.Optionally, sequencing of a nucleic acid template is monitored and thecontacting with the subset of nucleotides occurs upon detection ofphasing. See also for example, U.S. Pat. No. 8,236,532, which isincorporated herein by reference in its entirety.

Optionally, the sequencing reaction involves a plurality of templatenucleic acids, polymerases and/or nucleotides, wherein a plurality ofclosed-complexes is monitored. Clonally amplified template nucleic acidsmay be sequenced together wherein the clones are localized in closeproximity to allow for enhanced monitoring during sequencing.Optionally, the formation of a closed-complex ensures the synchronicityof base extension across a plurality of clonally amplified templatenucleic acids. The synchronicity of base extension allows for theaddition of only one base per sequencing cycle.

EXAMPLES

The following Example demonstrates how monitored destabilization ofternary complexes can be used in a sequencing-by-binding procedure.Ternary complexes were prepared using a primed template nucleic acidmolecule, a polymerase, and a plurality of nucleotides. Wash steps thatprogressively omitted nucleotides, one at a time, were used to identifycognate and non-cognate nucleotides without incorporation of anynucleotide into the primer. In this Example, polymerase was omitted fromthe wash buffer. Optionally, polymerase can be included in the washbuffer with similarly good results. Identification of cognate andnon-cognate nucleotides was based on assessment of formation and/ormaintenance of ternary complexes. A single incorporation step employingreversible terminator nucleotides facilitated single nucleotideincorporation. A first polymerase was used for conducting theexamination step with native nucleotides, and a second polymerase wasused in the incorporation step. The reversible terminator moiety of theblocked primer was removed by chemical treatment prior to the nextexamination step. All steps were repeated in a cyclical fashion.

Example 1 describes a procedure wherein cognate nucleotides of ternarycomplexes were identified by dissociation of those ternary complexes,without incorporation of any nucleotide into the primer of the primedtemplate nucleic acid. More particularly, ternary complexes weredestabilized when washed with a buffer that did not include the cognatenucleotide.

Example 1 Sequencing-By-Binding Using Monitored Dissociation of aTernary Complex

A FORTEBIO® (Menlo Park, Calif.) Octet instrument employing biolayerinterferometry to measure binding reactions at the surface of a fiberoptic tip was used in a multiwell plate format to illustrate thesequencing technique. Template strands biotinylated at their 5′-endswere used to immobilize primed template nucleic acid onto fiber optictips functionalized with streptavidin (SA) according to standardprocedures. Four different template sequences (i.e., wildtype, G12C,G12R, and G13D) were used to demonstrate interrogation of mutations incodons 12 and 13 of the KRAS sequence. Template sequences and targetswere selected to exemplify detection of each of four differentnucleotides by the procedure. Two different positions in the wildtype(WT) sequence were used for making comparisons. Relevant sequences wereas follows, where underlining identifies the base position beinginterrogated.

Codon 12 Wildtype GGT G12C TGT G12R CGT Codon 13 Wildtype GGC G13D GAC

Tips were washed in a buffered solution that included 30 mM Tris (pH8.0), 220 mM KCl, 160 mM potassium glutamate, and 0.01% Tween-20 beforecommencing the cycling protocol. A ternary complex was formed bycontacting the immobilized primed template nucleic acid with a bufferedsolution that included a polymerase and the combination of four nativedNTPs (dTTP, dGTP, dCTP, dATP), each of the dNTPs being present at aconcentration of 100 μM, for a period of about 30-100 seconds at 37° C.Polymerases used in the procedure were either Bst 2.0 (NEB; Ipswich,Mass.) at 360 U/ml, or Bsu DNA polymerase large fragment (New EnglandBioLabs; Ipswich, Mass.) at 136 U/ml. The solution used for preparingternary complexes further included 30 mM Tris-HCl (pH 8.0), 220 mM KCl,160 mM potassium glutamate, 0.01% Tween-20, 1 mM β-mercaptoethanol, and2 mM SrCl₂.

Cognate and non-cognate nucleotides were identified by observing thedissociation of a ternary complex following a series of wash steps. Allwash solutions used in the procedure included 30 mM Tris-HCl (pH 8.0),220 mM KCl, 160 mM potassium glutamate, 2 mM SrCl₂, 0.01% Tween-20, 1 mMβ-mercaptoethanol. Nucleotides, when present, were included atconcentrations of 100 μM each. Tips were first washed for 5-20 secondsusing a buffered solution that included three dNTPs (dGTP, dCTP, anddATP) while omitting one dNTP (dTTP) from the collection used to producethe ternary complex. Tips were next washed for 5-20 seconds in abuffered solution that included two dNTPs (dCTP, dATP) while omittingone dNTP (dGTP) from the collection used in the previous wash. Tips werenext washed for 5-20 seconds in a buffered solution that included onedNTP (dATP) while omitting one dNTP (dCTP) from the collection used inthe previous wash. Tips were finally washed for 5-20 seconds in abuffered solution that did not include any dNTP, again being consistentwith the pattern of omitting the single nucleotide (dATP) that had beenincluded in the previous wash step. While not used in this procedure,the initial wash step optionally can employ the complete set ofnucleotides used for preparing the ternary complex (e.g., all four dNTPsin this Example).

Results presented in FIGS. 1 and 2 illustrate sequencing runs carriedout using the protocol described above. FIG. 1 shows examination tracesfor the nucleotide mutation at codon 12 (GGT), which ordinarily encodesglycine (WT). The first G in this codon can be mutated to T or C, whichresults in codons encoding cysteine (G12C) or arginine (G12R),respectively. Ternary complexes were first generated in the presence ofall 4 dNTPs (dTTP, dGTP, dCTP, dATP) using each different primedtemplate nucleic acid. Complexes were subsequently washed using a bufferthat included three dNTPs (dGTP, dCTP, dATP), but not dTTP. Ternarycomplexes dissociated and the binding signal was lost when the cognatebase was a T (as in G12C). As shown in FIG. 1, the observed dissociationwas specific for omission of the cognate nucleotide, meaning that otherternary complexes (e.g., including primed template nucleic acids fortemplates G12R and WT) remained intact when the wash buffer includeddGTP, dCTP, and dATP. When nucleotides present in the next wash werelimited to dCTP and dATP, there was substantially no further reductionin the binding signal for the G12C trial, because the ternary complexalready had dissociated. However, the ternary complex that included theWT primed template nucleic acid dissociated and the binding signal waslost since dGTP had been omitted from the wash buffer, and since dGTPwas the cognate nucleotide in that example. Again the cognate nucleotideof the WT template was identified by dissociation of the ternary complexthat included that nucleotide. When the wash buffer included only dATPand not dCTP, the ternary complex that included G12R dissociated,thereby identifying dCTP as the cognate nucleotide for that complex.Finally, a wash buffer that did not include any of the four nucleotidesshowed no further signal reductions for any of the three templates shownin FIG. 1. FIG. 2 shows examination traces for codon 13 (GGC), whichordinarily encodes glycine (WT), but which can be mutated to encodeaspartate (G13D) by changing the second position of the codon to an A(i.e., GAC). When the cognate dGTP nucleotide was omitted from the washbuffer, ternary complexes that included the WT primed template nucleicacid dissociated and binding signal was lost. Likewise, elimination ofthe cognate dATP nucleotide from the wash buffer (i.e., the final washbuffer that did not include any dNTP) led to dissociation of ternarycomplexes that included the G13D primed template nucleic acid.

Taken together, the results presented in FIGS. 1 and 2 confirmed that anucleotide that included a base complementary to the next base of atemplate strand immediately downstream of the primer in a primedtemplate nucleic acid could be identified by a process that involvedmonitoring dissociation of ternary complexes. More specifically,dissociation of a ternary complex indicated that the cognate nucleotidehad been eliminated from the complex. On the other hand, persistence ofa ternary complex in the absence of a test nucleotide indicated the testnucleotide was not the cognate nucleotide. Notably, monitoringoptionally can involve assessment of binding signals at the start andfinish of the individual steps (i.e., endpoint monitoring).

Described below is an approach employing initial formation ofnucleotide-independent binary complexes, followed by addition ofnucleotides to produce ternary complexes. Complexes formed in theprocedure were subjected to a series of wash steps, during which timeternary complex maintenance and dissociation was monitored. For example,binary complexes that included primed template nucleic acid andpolymerase can be contacted with a reaction mixture including fournative nucleotides. Serial washes (e.g., using 3, 2, 1, and 0nucleotides) can be used with monitoring to establish whennucleotide-specific ternary complexes dissociate. The nucleotiderequired to maintain integrity of the ternary complex (i.e., thenucleotide that, when removed, causes dissociation of the complex)corresponds to the cognate nucleotide. The following Example illustratesthe technique using binding of two nucleotides, rather than fournucleotides to form ternary complexes. Two sets of two nucleotides wererequired to test the full complement of four dNTPs. By this approach,only one of the two sets of two nucleotides for each primed templatenucleic acid formed a ternary complex. The other set of two nucleotides,which did not include the cognate nucleotide, did nothing to modify thebinary complex. Results presented below evidenced the distinctionbetween sets of nucleotides capable of forming ternary complexes, andsets of nucleotides that did not alter the preformednucleotide-independent binary complexes.

Example 2 describes a procedure wherein cognate nucleotidescorresponding to each of dATP, dTTP, dGTP, and dCTP were identified by aprocess involving initial formation of binary complexes, followed bymonitoring of formation and/or dissociation of ternary complexes.Notably, failure to detect a ternary complex in the presence of aplurality of nucleotides indicated that none of the nucleotides amongthe plurality corresponded to the cognate nucleotide.

Example 2 Preliminary Formation of Binary Complexes Followed byFormation and Dissociation of Ternary Complexes Identifies Cognate andNon-Cognate Nucleotides

A FORTEBIO® (Menlo Park, Calif.) Octet instrument employing biolayerinterferometry to measure binding reactions at the surface of a fiberoptic tip was used in a multiwell plate format to illustrate thesequencing technique. Template strands biotinylated at their 5′-endswere used to immobilize primed template nucleic acid onto fiber optictips functionalized with streptavidin (SA) according to standardprocedures. Four different template sequences (i.e., wildtype, G12C,G12R, and G13D) were used to demonstrate interrogation of mutations incodons 12 and 13 of the KRAS sequence. Template sequences and targetswere selected to exemplify detection of each of four differentnucleotides by the invented procedure. Two different positions in thewildtype (WT) sequence were used for making comparisons. Relevantsequences were as follows, where underlining identifies the baseposition being interrogated.

Codon 12 Wildtype GGT G12C TGT G12R CGT Codon 13 Wildtype GGT G13D GAC

Tips were washed in a buffered solution containing 200 mM KCl, 160 mMpotassium glutamate, and 0.01% Tween-20 before commencing the cyclingprotocol. Nucleotide-independent binary complexes were formed bycontacting tips harboring immobilized primed template nucleic acid witha solution that included 30 mM Tris-HCl (pH 8.0), 220 mM KCl, 160 mMpotassium glutamate, 2 mM SrCl₂, 0.01% Tween-20, 1 mM β-mercaptoethanol,and Bst 2.0 (NEB; Ipswich, Mass.) DNA polymerase, but that did notinclude any added nucleotide. Next, the enzyme-containing solution wasreplaced with a second reaction mixture that included dTTP and dGTP,each at a concentration of 100 μM, to permit nucleotide binding andternary complex formation if either nucleotide corresponded to thecognate nucleotide. The nucleotide-containing solution further included30 mM Tris-HCl (pH 8.0), 220 mM KCl, 160 mM potassium glutamate, 0.01%Tween-20, 1 mM β-mercaptoethanol, and 2 mM SrCl₂. An optional wash stepwas conducted using a buffer that included all (i.e., two) of thenucleotides used in the nucleotide binding step. Next, tips were washedwith a buffer that omitted one of the nucleotides (dTTP) from theprevious wash step. Any polymerase and nucleotide remaining in ternarycomplexes were removed with 30 mM Tris-HCl (pH 8.0), 320 mM KCl, 20 mMEDTA, 0.01% Tween-20, 1 mM β-mercaptoethanol. Optionally, an additionalwash step could have been included immediately before the wash thatremoved nucleotide and ternary complexes to permit dissociation ofresidual ternary complexes by the same mechanism used for nucleotideinterrogation (i.e., omission of cognate nucleotide from the buffer usedfor examination). The process was repeated using the remaining twonucleotides (i.e., dCTP and dATP) in place of the first set ofnucleotides, and using appropriate wash buffers according to the cyclingintervals indicated in FIGS. 3A-3D.

Results of the procedure are illustrated in FIGS. 3A-3D. In allinstances, results illustrate a two-part procedure wherein binarycomplexes were formed before contacting nucleotides. Binary complexeswere contacted with the first two nucleotides (dTTP and dGTP) toinvestigate possible ternary complex formation. Complexes were thensubjected to wash steps that progressively omitted one of thenucleotides being tested for the ability to promote ternary complexformation. The second part of the procedure repeated the first partwhile substituting the remaining two nucleotides (dCTP and dATP) inplace of the first two nucleotides.

FIG. 3A shows results obtained in a system wherein the next correctnucleotide was dATP. Binary complexes formed between the primed templatenucleic acid and polymerase were contacted with a solution that includeddTTP and dGTP, but did not include polymerase to maintain binarycomplexes in the absence of cognate nucleotide. Binding signal decreasedimmediately and steadily after washes that included dTTP and dGTP, ordGTP alone. The failure to increase or even maintain the binding signalindicated that a ternary complex did not form. The absence of ternarycomplex formation indicated that neither dTTP nor dGTP was the cognatenucleotide. The second part of the procedure involved formation ofbinary complexes, followed by contact with dCTP and dATP, and washingwith buffers that included either dCTP and dATP, or dATP alone. Theincrease in signal observed following contact with the combination ofdCTP and dATP indicated that a ternary complex had formed, and that oneof dCTP and dATP was the cognate nucleotide. The fact that a bindingcomplex was maintained in the absence of dCTP, and in the presence ofdATP indicated that dCTP was the non-cognate nucleotide and that dATPwas the cognate nucleotide.

FIG. 3B shows results obtained in a system wherein the next correctnucleotide was dTTP. Binary complexes formed between the primed templatenucleic acid and polymerase were contacted with a solution that includeddTTP and dGTP, but did not include polymerase needed to maintain binarycomplexes in the absence of cognate nucleotide. Binding signal increasedslightly upon contact with the solution that included dTTP and dGTP,thereby indicating that one of dTTP and dGTP was the cognate nucleotide.Binding signal was maintained substantially constant until dTTP wasomitted from the wash buffer, at which point complexes dissociated. Thisindicated dTTP was the cognate nucleotide. In the second part of theprocedure, wherein dCTP and dATP were tested for possible ternarycomplex formation, the binding signal decreased as soon as polymerasewas withdrawn (i.e., contact with a solution including dCTP and dATP,but not including polymerase). Absent substantial maintenance of thebinding signal, or a positive slope of a line joining the first and lastmeasured data points of the interval corresponding to contact with dCTPand dATP (as would characterize a signal increase), it was confirmedthat neither dCTP nor dATP was the cognate nucleotide.

FIG. 3C shows results obtained in a system wherein the next correctnucleotide was dGTP. Binary complexes formed between the primed templatenucleic acid and polymerase were contacted with a solution that includeddTTP and dGTP, but did not include polymerase needed to maintain binarycomplexes in the absence of cognate nucleotide. Binding signal increasedslightly upon contact with the solution that included dTTP and dGTP,thereby indicating that one of dTTP and dGTP was the cognate nucleotide.Binding signal was maintained substantially constant as long as dGTP wasincluded in the wash buffer, thereby indicating that dGTP was thecognate nucleotide. In the second part of the procedure, wherein dCTPand dATP were tested for possible ternary complex formation, the bindingsignal decreased as soon as polymerase was withdrawn (i.e., contact witha solution including dCTP and dATP, but not including polymerase).Absent substantial maintenance of the binding signal, or a positiveslope of a line joining the first and last measured data points of theinterval corresponding to contact with dCTP and dATP, it was confirmedthat neither dCTP nor dATP was the cognate nucleotide.

FIG. 3D shows results obtained in a system wherein the next correctnucleotide was dCTP. Binary complexes formed between the primed templatenucleic acid and polymerase were contacted with a solution that includeddTTP and dGTP, but did not include the polymerase needed to maintainbinary complexes in the absence of cognate nucleotide. Binding signaldecreased immediately and steadily after washes that included dTTP anddGTP, or dGTP alone. Absent substantial maintenance of the bindingsignal, or a positive slope of a line joining the first and lastmeasured data points of the interval corresponding to contact with dTTPand dGTP, it was confirmed that neither dTTP nor dGTP was the cognatenucleotide. The second part of the procedure involved formation ofbinary complexes followed by contact with dCTP and dATP, at which pointa positive slope was observed between the first and last points of theinterval that involved contact with dCTP and dATP, thereby indicatingthat one of dCTP and dATP was the cognate nucleotide. Binding signalmaintained substantially constant until dCTP was omitted from the washbuffer, at which point the binding signal decreased to indicate loss ofternary complexes. Accordingly, dCTP was the cognate nucleotide.

The following Examples illustrate examination steps that employedcatalytic amounts of Mg²⁺ ion in combination with a primer having a3′-blocking group. The blocking group of the primer was removed aftermeasuring binding of the primed template nucleic acid molecule topolymerase in the presence of each different native nucleotide. Themeasured binding was sufficient to identify which of the nucleotidesrepresented the cognate nucleotide for a particular position. In thenext step of the workflow, a reversible terminator nucleotide wasincorporated without any intervening examination step (i.e., withoutintervening binding, detection or identification of any nucleotide).Notably, examination and incorporation steps were carried out using twodifferent polymerase enzymes. Optionally, a single polymerase, such asthe 3PDX polymerase disclosed in U.S. Pat. No. 8,703,461 for the purposeof interrogating nucleotide analogs and incorporating reversibleterminator nucleotides, also may be used.

Example 3 describes examination of a primed template nucleic acidmolecule, where the primer strand was blocked from extension at its3′-end. The examination step (i.e., involving measuring interactionbetween the primed template nucleic acid molecule, the polymerase, and atest nucleotide) was conducted in the presence of a catalytic metal ionwith the intention of enhancing discriminatory activity of thepolymerase enzyme. Results presented below demonstrated efficientidentification of the next correct nucleotide, and even the followingnext correct nucleotide.

Example 3 Examination of a Primed Template Nucleic Acid Molecule Havinga 3′-Blocked Primer in the Presence of Catalytic Concentrations ofMagnesium Ion

A FORTEBIO® (Menlo Park, Calif.) Octet instrument employing biolayerinterferometry to measure binding reactions at the surface of a fiberoptic tip was used in a multiwell plate format to illustrate thesequencing technique. Template strands biotinylated at their 5′-endswere used to immobilize primed template nucleic acid molecule onto fiberoptic tips functionalized with streptavidin (SA) according to standardprocedures. A 3′-blocked primer was prepared by incorporating a cognatereversible terminator nucleotide that included a 3′-ONH₂ blocking group,using Therminator DNA polymerase (New England BioLabs Inc.; Ipswich,Mass.) according to the manufacturer's instructions. A description ofthe reversible terminator nucleotide can be found in U.S. Pat. No.7,544,794, the disclosure of which is incorporated by reference. Bindingof incoming nucleotides was investigated using 68 units/mL of Bsu DNApolymerase (New England BioLabs Inc.; Ipswich, Mass.) in a buffer thatfurther included 30 mM Tris (pH 8.0), 220 mM KCl, 160 mM potassiumglutamate, 1 mM MgCl₂, 0.01% Tween-20, and 1 mM β-mercaptoethanol.Ternary complex formation indicating cognate nucleotide binding wasinvestigated by contacting the primed template nucleic acid moleculehaving the 3′-blocked primer with the Bsu DNA polymerase and one of fournative dNTP nucleotides (dATP, dGTP, dCTP, and dTTP) for a period of 20seconds. Each of the different nucleotides was used at a concentrationof 100 μM during the examination procedure. Thereafter, biosensors werewashed with a solution that included 20 mM EDTA for 25 seconds tochelate magnesium ions. The biosensors were then equilibrated withregeneration buffer that included 30 mM Tris (pH 8.0), 220 mM KCl, 160mM potassium glutamate, 1 mM MgCl₂, 0.01% Tween-20, 1 mMβ-mercaptoethanol. The same steps were repeated for the remainingnucleotides in sequence until collecting all binding curves for all fourdNTPs. After completing examination of the different nucleotides, andacquiring measurement data for identifying the next correct nucleotide,the biosensor was transferred into a cleavage buffer solution (1 Msodium acetate pH 4.5 and 500 mM NaNO₂) for 60 seconds to remove theblocking group from the 3′-end of the primer. Biosensors were nextequilibrated with a regeneration buffer (20 mM Tris pH 8.0, 10 mM KCl,and 0.01% Tween-20). Correct nucleotide was subsequently incorporatedusing the Therminator polymerase at a concentration of 30 units/mL in abuffer that included 20 mM Tris (pH 8.8), 10 mM ammonium sulfate, 10 mMKCl, 2 mM MgCl₂, 0.1% Triton-X-100, and all four reversible terminatornucleotides at a concentration of 100 μM each. All buffers were preparedwith HPLC grade water and the incorporation buffer included HPLC waterwith 10 wt % OH—NH₂. The incorporation step was carried out for 60seconds, after which time the bound polymerase was washed away from thebiosensor with 20 mM EDTA for 5 seconds before commencing the nextexamination cycle, as described above.

FIG. 4 shows the traces for all four nucleotides followed by thecleavage traces and incorporation traces, as discussed above. Theexpected base sequence in this example was GAC. As described above, a3′-ONH₂ blocked primer was first formed by incorporating a reversibleterminator using the Therminator polymerase. Next, for each cycle ofexamination the blocked primed template nucleic acid molecule wascontacted with polymerase and a different nucleotide (dATP or dTTP ordGTP or dCTP) in the presence of catalytic magnesium ions for 20seconds. High binding signals were observed if the examined nucleotideincluded the complementary base to the next base of the template strand.In addition to this peak, a second-high binding signal was also observedfor the second correct complementary base to the second next base of thetemplate strand. After all four nucleotides had been examined, acleavage reaction removed the 3′ blocking group from the primer. Afterremoving the cleavage reagent with two wash steps (corresponding to thetwo steps with progressively reduced binding signals immediatelyfollowing the cleavage step), a single incorporation reaction wascarried out to add the next reversible terminator nucleotide. Theprocedure can be used for identifying the next correct nucleotide (nextincoming nucleotide at the n+1 position), and can be repeated aplurality of times to determine the sequence of the template nucleicacid. As well, the results showed how the correct nucleotide at the n+2position also could be determined. This observation was reproduced forall the positions in the sequence. Optionally, serial incorporation oftwo reversible terminators can be carried out without interveningexamination steps using different types of nucleotides (i.e., other thanreversible terminators) to speed the process of sequence determination.

The foregoing procedure employed a plurality of examination reactions toobtain the information needed for identifying a cognate nucleotidebefore conducting an incorporation reaction using reversible terminatornucleotides. Generally speaking, data processing for base calling neednot be contemporaneous with the examination and incorporation reactionsusing this approach. Optionally, base calling algorithms can employrecorded measurement data acquired during the examination steps.Further, it is to be understood that while examination and incorporationsteps in the illustrated protocol used two different enzymes todemonstrate procedural flexibility, these steps optionally can becarried out using the same polymerase enzyme. Still further, thereversibly blocked primer employed in the examination step permitted useof a catalytic metal ion during that step. Optionally, however,non-catalytic metal ions, or mixtures of non-catalytic and catalyticmetal ions, can be substituted in place of the catalytic metal ions whenusing a primed template nucleic acid molecule having a 3′-blockedprimer.

Example 4 describes a dissociation-based sequencing protocol, wherein anincorporation reaction that was conducted after a plurality ofexamination reactions facilitated rapid nucleotide identification.Interaction of a polymerase and a primed template nucleic acid moleculehaving a reversibly blocked primer (referred to herein as a “blockedprimed template nucleic acid molecule”) were measured or monitoredcontinuously to document the binding reaction mechanism. Periodic (e.g.,end-point) measurements may be simpler to execute, and can be used toobtain similarly good results. After measuring interaction of apolymerase with the blocked primed template nucleic acid molecule todetermine whether or not a nucleotide under investigation was thecognate nucleotide for a particular position, the reversible terminatormoiety of the blocked primed template nucleic acid molecule was removedbefore incorporating the next reversible terminator nucleotide. Theprimed template nucleic acid molecule having a free 3′-hydroxyl moietynever contacted any nucleotide other than those comprising reversibleterminator moieties. As in the preceding Example, incorporation ofreversible terminator nucleotides was performed using a polymerasedifferent from the one used for examining transient binding ofnucleotides (i.e., investigating ternary complex formation). However, asdiscussed above, a single polymerase enzyme may also be used to performboth of these functions in a simplified procedure.

Example 4 Dissociation-Based Sequencing-by-Binding Employing Examinationof Reversibly Blocked Primers in the Presence of CatalyticConcentrations of Magnesium Ions

A FORTEBIO® (Menlo Park, Calif.) Octet instrument employing biolayerinterferometry to measure binding reactions at the surface of a fiberoptic tip was used in a multiwell plate format to illustrate thesequencing technique. Template strands biotinylated at their 5′-endswere used to immobilize primed template nucleic acid molecule onto fiberoptic tips functionalized with streptavidin (SA) according to standardprocedures. A 3′-blocked primer was prepared by incorporating a cognatereversible terminator nucleotide that included a 3′-ONH₂ blocking group,using Therminator DNA polymerase (New England BioLabs Inc.; Ipswich,Mass.) according to the manufacturer's instructions. A description ofthe reversible terminator nucleotide can be found in U.S. Pat. No.7,544,794, the disclosure of which is incorporated by reference. Bindingof incoming nucleotides was investigated using either Bsu DNA polymeraseor Bst 2.0 DNA polymerase, both of which were obtained from New EnglandBioLabs Inc. (Ipswich, Mass.). The Bsu DNA polymerase was used at aconcentration of 400 U/mL, while the Bst 2.0 DNA polymerase was used ata concentration of 600 U/mL, each in a buffer that further included 30mM Tris (pH 8.0), 220 mM KCl, 160 mM potassium glutamate, 1 mM MgCl₂,0.01% Tween-20, and 1 mM β-mercaptoethanol. Sensor tips having blockedprimed template nucleic acid molecules immobilized thereon wereinitially contacted with polymerase in the absence of any nucleotide fora period of 20 seconds. Next, sensor tips were removed from thepolymerase-containing solution and contacted for a period of 10 secondswith a solution that included the polymerase and a paired set of twonucleotides (e.g., dTTP and dATP), each of the nucleotides being presentat a concentration of 100 μM. Cognate nucleotide corresponding to thenext correct nucleotide participated in formation of a ternary complexduring this step. Next, sensor tips were washed for 30 seconds with asolution that included 20 mM EDTA to chelate Mg²⁺ ions and removepolymerase and nucleotide from the sensor tip. Next, sensor tips wereremoved from the EDTA-containing solution and contacted for a period of10 seconds with a solution that included the polymerase and a secondpaired set of two nucleotides (e.g., dCTP and dGTP), each of thenucleotides being present at a concentration of 100 μM. Again, cognatenucleotide corresponding to the next correct nucleotide participated information of a ternary complex during this step. Next, sensor tips werewashed for 30 seconds with a solution that included 20 mM EDTA tochelate Mg²⁺ ions and remove polymerase and nucleotide from the sensortip. At this point all binding results required for identifying the nextcorrect nucleotide had been acquired, without performing anyincorporation reaction. The biosensor was then transferred into acleavage buffer solution (1 M sodium acetate (pH 5.5) and 500 mM NaNO₂)for 60 seconds to remove the blocking group from the 3′-end of theprimer. Biosensors were next equilibrated with a regeneration buffer (20mM Tris (pH 8.0), 10 mM KCl, and 0.01% Tween-20). Reversible terminatornucleotides corresponding to next correct bases were incorporated intothe primer using the Therminator polymerase (New England BioLabs Inc.)at 30 units/mL in a buffer that included 20 mM Tris (pH 8.8), 10 mM(NH₄)₂SO₄, 10 mM KCl, 5 mM MgCl₂, and 0.1% Triton-X-100, and furtherincluded all four reversible terminators at 100 μM concentrations each.All buffers were prepared with HPLC grade water and the incorporationbuffer included HPLC water with 1 wt % OH—NH₂. The incorporation stepwas allowed to proceed for 60 seconds. Polymerase was removed by washingwith 20 mM EDTA for 30 seconds before the new cycles of polymerasebinding were begun.

FIG. 5A illustrates three complete cycles of reversible terminatorincorporation/examination/removal of the reversible terminator moiety,where Bsu DNA polymerase was used to conduct the examination steps.Numbers have been assigned to each step within the different cycles forconvenience. Referring now to cycle 1 (representative of the entireprocedure), step 1 corresponded to incorporation of a reversibleterminator moiety into the primer. The very high signal observed in thisstep was not informative with respect to nucleotide identity. Steps 2and 3 corresponded respectively to an EDTA wash step (to remove anybound polymerase), and a regenerating buffer wash step (to remove EDTAand adjust buffer conditions). Polymerase binding in the absence ofnucleotide in step 4 increased the binding signal. The signal wasfurther increased in step 5, after the blocked primed template nucleicacid contacted a reaction mixture that included dATP and dTTP inaddition to the polymerase from step 4. The substantial increase inbinding signal observed during step 5 reflected ternary complexformation, and indicated that one of dATP and dTTP was the cognatenucleotide for the position being interrogated. Washing with a solutionthat included, in addition to the polymerase of step 4, dATP but notdTTP led to a substantial reduction of the binding signal in step 6.This indicated that the ternary complex became unstable and wasdisrupted in the absence of dTTP. Accordingly, dTTP was identified asthe next correct nucleotide. Steps 7 and 8 corresponded respectively toanother EDTA wash step (to remove any bound polymerase) and anotherregenerating buffer wash step (to remove EDTA and adjust bufferconditions). In step 9, contacting the blocked primed template nucleicacid molecule with the same polymerase used in step 4, again in theabsence of nucleotide, increased the binding signal. The binding signaldid not substantially increase in step 10, after the blocked primedtemplate nucleic acid molecule contacted a solution that included dGTPand dCTP in addition to the polymerase of step 4. The absence of asubstantial increase in binding signal indicated that a ternary complexdid not form. Accordingly, neither of dGTP and dCTP was the cognatenucleotide for that position. In step 11, washing with a solution thatincluded the polymerase of step 4 and dGTP but not dCTP did notsubstantially change the binding signal, as expected (i.e., becauseneither dGTP nor dCTP was the cognate nucleotide for this position).Another EDTA wash in step 12 removed any bound polymerase, and asubsequent wash changed buffer conditions in step 13. Cleavage of thereversible terminator moiety in step 14 revealed a free 3′-OH group onthe primer that was available to participate in phosphodiester bondformation. Two wash steps in steps 15 and 16 prepared the primedtemplate nucleic acid molecule to receive the next reversible terminatornucleotide by an enzymatic incorporation reaction.

FIG. 5B illustrates three complete cycles of reversible terminatorincorporation/examination/removal of the reversible terminator moiety,where Bst 2.0 DNA polymerase was used to conduct the examination steps.The results were consistent with those presented in FIG. 5A, that wereobtained using a different DNA polymerase in the examination steps.

Disclosed above are materials, compositions, and components that can beused for, can be used in conjunction with, can be used in preparationfor, or are products of the disclosed methods and compositions. It is tobe understood that when combinations, subsets, interactions, groups,etc. of these materials are disclosed, and that while specific referenceof each various individual and collective combinations and permutationsof these compounds may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a method is disclosedand discussed and a number of modifications that can be made to a numberof molecules including the method are discussed, each and everycombination and permutation of the method, and the modifications thatare possible are specifically contemplated unless specifically indicatedto the contrary. Likewise, any subset or combination of these is alsospecifically contemplated and disclosed. This concept applies to allaspects of this disclosure, including steps in methods using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed, it is understood that each of these additionalsteps can be performed with any specific method steps or combination ofmethod steps of the disclosed methods, and that each such combination orsubset of combinations is specifically contemplated and should beconsidered disclosed.

Publications cited herein, and the material for which they are cited,are hereby specifically incorporated by reference in their entireties.All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that the headings used herein are fororganizational purposes only and are not meant to limit the descriptionor claims.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the claims.

What is claimed is:
 1. A method of identifying a nucleotide comprising abase complementary to the next base of a template strand immediatelydownstream of a primer in a primed template nucleic acid molecule, saidmethod comprising the steps of: (a) providing a blocked primed templatenucleic acid molecule comprising a reversible terminator moiety thatprecludes the 3′-terminus of the blocked primed template nucleic acidmolecule from participating in phosphodiester bond formation; (b)contacting the blocked primed template nucleic acid molecule with afirst reaction mixture that comprises a polymerase, and a plurality ofdifferent nucleotide molecules, whereby a stabilized ternary complexforms, said stabilized ternary complex comprising one of the pluralityof different nucleotide molecules; (c) contacting the stabilized ternarycomplex with a second reaction mixture that comprises at least one ofthe different nucleotide molecules and that does not comprise a firstnucleotide molecule of the plurality of different nucleotide molecules;(d) monitoring interaction of the polymerase and the blocked primedtemplate nucleic acid molecule in contact with the second reactionmixture to detect any of the stabilized ternary complex remaining afterstep (c); and (e) identifying the nucleotide that comprises the basecomplementary to the next base of the template strand using results fromstep (d).
 2. The method of claim 1, further comprising the step of: (f)removing the reversible terminator moiety from the blocked primedtemplate nucleic acid molecule after step (d).
 3. The method of claim 2,wherein step (e) comprises determining either that: (i) the firstnucleotide molecule in step (c) comprises the base complementary to thenext base of the template strand if the stabilized ternary complexdissociates in step (d), or (ii) the first nucleotide molecule in step(c) does not comprise the base complementary to the next base of thetemplate strand if the stabilized ternary complex is retained in step(d).
 4. The method of claim 3, wherein the polymerase of the firstreaction mixture comprises an exogenous fluorescent label.
 5. The methodof claim 3, wherein the plurality of different nucleotide molecules inthe first reaction mixture is either a plurality of different nativenucleotide molecules, or a plurality of different fluorescently labelednucleotide molecules.
 6. The method of claim 5, wherein the firstreaction mixture further comprises a catalytic metal ion.
 7. The methodof claim 5, wherein the first reaction mixture does not comprisenon-catalytic metal ions that inhibit phosphodiester bond formation bythe polymerase of the first reaction mixture, and wherein the firstreaction mixture further comprises a catalytic metal ion.
 8. The methodof claim 3, wherein step (a) comprises incorporating, with a polymerase,a reversible terminator nucleotide at the 3′-end of the primer of theprimed template nucleic acid molecule, whereby there is produced theblocked primed template nucleic acid molecule comprising the reversibleterminator moiety that precludes the 3′-terminus of the blocked primedtemplate nucleic acid molecule from participating in phosphodiester bondformation.
 9. The method of claim 8, further comprising, after step (a)and before step (b), a step of contacting the blocked primed templatenucleic acid molecule with the polymerase of the first reaction mixturein the absence the plurality of different nucleotide molecules.
 10. Themethod of claim 8, wherein the second reaction mixture comprises thesame polymerase that is present in the first reaction mixture.
 11. Themethod of claim 8, further comprising, after step (a) and before step(b), the step of contacting the blocked primed template nucleic acidmolecule with the polymerase of the first reaction mixture in theabsence of the plurality of different nucleotide molecules.
 12. Themethod of claim 11, wherein the first reaction mixture further comprisesa catalytic metal ion.
 13. The method of claim 11, wherein the secondreaction mixture comprises the same polymerase that is present in thefirst reaction mixture.
 14. The method of claim 8, further comprisingrepeating steps (b)-(e) a plurality of times.
 15. The method of claim14, wherein the polymerase used in step (a) and the polymerase of thefirst reaction mixture in step (b) are different types of polymeraseenzymes.
 16. The method of claim 14, wherein the polymerase of the firstreaction mixture comprises an exogenous fluorescent label.
 17. Themethod of claim 14, wherein the plurality of different nucleotidemolecules in the first reaction mixture is either a plurality ofdifferent native nucleotide molecules, or a plurality of differentfluorescently labeled nucleotide molecules.
 18. The method of claim 14,wherein the first reaction mixture further comprises a catalytic metalion.
 19. The method of claim 14, wherein the first reaction mixture doesnot comprise non-catalytic metal ions that inhibit phosphodiester bondformation by the polymerase of the first reaction mixture.
 20. Themethod of claim 14, wherein step (f) is performed before step (e). 21.The method of claim 14, wherein the second reaction mixture comprisesthe same polymerase that is present in the first reaction mixture.
 22. Amethod of identifying a nucleotide comprising a base complementary tothe next base of a template strand immediately downstream of a primer ina primed template nucleic acid molecule, said method comprising: (a)providing the primed template nucleic acid molecule; (b) contacting theprimed template nucleic acid molecule with a first reaction mixture thatcomprises a polymerase and a plurality of different nucleotidemolecules, whereby a stabilized ternary complex forms, said stabilizedternary complex comprising one of the plurality of different nucleotidemolecules; (c) contacting the primed template nucleic acid molecule,after step (b), with a second reaction mixture that comprises at leastone of the different nucleotide molecules and that does not comprise afirst nucleotide molecule of the plurality of different nucleotidemolecules; (d) monitoring interaction of the polymerase and the primedtemplate nucleic acid molecule in the second reaction mixture, withoutincorporating any nucleotide into the primer, to detect any of thestabilized ternary complex remaining after step (c); and (e) identifyingthe nucleotide that comprises the base complementary to the next base ofthe template strand using results from step (d).
 23. A method ofidentifying a nucleotide comprising a base complementary to the nextbase of a template strand immediately downstream of a primer in a primedtemplate nucleic acid molecule, said method comprising: (a) providingthe primed template nucleic acid molecule; (b) contacting the primedtemplate nucleic acid molecule with a first reaction mixture thatcomprises a polymerase, but does not comprise any nucleotide, whereby abinary complex forms; (c) contacting the binary complex with a secondreaction mixture that comprises a plurality of different nucleotidemolecules, whereby a stabilized ternary complex forms if one of theplurality of different nucleotide molecules comprises the basecomplementary to the next base of the template strand; (d) detecting,without incorporating any nucleotide into the primer, any of thestabilized ternary complex that may have formed; (e) contacting theprimed template nucleic acid molecule, after step (d), with a thirdreaction mixture that comprises at least one of the different nucleotidemolecules and that does not comprise a first nucleotide molecule of theplurality of different nucleotide molecules; (f) detecting, withoutincorporating any nucleotide into the primer, any of the stabilizedternary complex remaining after step (e); and (g) identifying thenucleotide that comprises the base complementary to the next base of thetemplate strand using results from both of detecting steps (d) and (f).